Composition comprised of antigen linked to a tnf superfamily ligand

ABSTRACT

The invention provides fusion proteins comprising antigens of infectious disease agents and cancer cells linked to multiple-trimer forms of TNF SuperFamily (TNFSF) ligands. The TNFSFs serve as vaccine adjuvants for increasing the immune response to the antigens. In particular, a fusion polypeptide strand that self-assembles inside cells into a multiple-trimer form of CD40 ligand (CD40L, TNFSF5) is provided. Other similar fusion proteins are also disclosed. The fusion proteins can be delivered to a host as isolated proteins, as nucleic acids used directly in DNA vaccination or carried and expressed by a viral vector such as adenovirus. In addition to use as a vaccine to prevent or ameliorate disease caused by an infectious agent, compositions of the invention may be used for the treatment of ongoing infection or for cancer immunotherapy.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. AI068489awarded by the National Institutes of Health (NIH) of the United Statesof America. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to compositions useful forgenerating or enhancing an immune response against an antigen, tomethods for using the compositions, and to modified immune cells usefulin such methods.

BACKGROUND OF THE INVENTION

Industrial applications of vaccines: Vaccines are considered to be amongthe most cost-effective and health-preserving medical inventions everdeveloped. The rationale for vaccination is that pre-exposure of thehost to a vaccine against a given infectious agent can ameliorate orprevent disease should the vaccinated individual become exposed to thatagent at a later time. The gap in time between vaccination and possibleexposure requires “memory” on the part of the immune system. This memoryis embodied in the persistence of immune cells for years or even decadesafter vaccination. Creating vaccines that induce strong and lastingprotection is a difficult task, given our incomplete knowledge of theimmune system. Nevertheless, continuing advances in our understandingmake possible new approaches to vaccine design.

Vaccines against infectious agents: For microbial agents, many vaccinesin use are comprised of live attenuated or non-virulent strains of thedisease-causing microorganisms. Other vaccines are comprised of killedor otherwise inactivated microorganisms. Yet other vaccines utilizepurified components of pathogen lysates, such as surface carbohydratesor recombinant pathogen-derived proteins. Vaccines that utilize liveattenuated or inactivated pathogens typically yield a vigorous immuneresponse, but their use has limitations. For example, live vaccinestrains can sometimes mutate back into disease-causing variants,especially when administered to immunocompromised recipients. Moreover,many pathogens, particularly viruses, undergo continuous rapid mutationsin their genome, which allow them to escape immune responses toantigenically distinct vaccine strains.

Vaccines for the prevention or treatment of cancer: As the understandingof immunity has developed, it became clear that the immune system alsocontrols or attempts to control the development of malignancies (Dunn etal., 2002; 3(11):991-8). As a result, immunotherapy is now being used toeradicate or control certain human cancers. Some of the technology andconcepts of vaccines against infectious agents also apply to using theimmune system to fight cancers, both solid tumors and blood cancers suchas leukemia. Patients at risk for cancer, such as those infected bycancer-associated viruses like human papilloma virus (HPV), can beprotected from developing the particular cancer in question asexemplified by Gardasil® vaccination against human papilloma virus(HPV), which causes cervical cancer. Patients who already have cancer,such as prostate cancer, can also be helped by vaccination, asexemplified by the Provenge® vaccine which is an immunotherapy forprostate cancer.

CD8+ T cells can recognize conserved antigens in many infectious agentsand prevent disease: While these have been successful vaccines, therehave been major problems constructing vaccines against antigens fromrapidly mutating infectious agents such as influenza, HIV, andPlasmodium falciparum (a cause of malaria). In these cases and others,the infectious agent has surface protein(s) that can rapidly mutate toevade otherwise protective antibodies. Nevertheless, these agents alsohave relatively conserved and unchanging internal components asexemplified by nucleoprotein (NP) of influenza, Gag and Pol for HIV, andcircumsporozoite surface protein (CSP) for Plasmodium falciparum. Inthese cases, antibodies (which can only bind to the surface ofpathogens) are unable to bind to these more conserved and internalantigens. Instead, there is a well-established role for CD8+ T cells incontrolling or clearing such infectious agents—provided that a strongenough CD8+ T cell response can be generated. To cite just threeexamples: (1) Protection from disease caused by influenza can beachieved by high levels of CD8+ T cells against the conservednucleoprotein (NP) viral protein (Webster et al., Eur J Immunol. 1980;10(5):396-401; Slutter et al., J Immunol. 2013; 190(8):3854-8. PMCID:3622175.). (2) Strong CD8+ T cell responses against the Gag and Polproteins of simian immunodeficiency virus (SIV, a non-human primatemodel for HIV infection) can protect macaques from developing AIDS afterchallenge with SIV (Hansen et al., Nature. 2011; 473(7348):523-7. PMCID:3102768). (3) CD8+ T cells against Plasmodium falciparum antigens canprotect humans from malaria (Epstein et al., Science. 2011;334(6055):475-80). Thus, there is an urgent and largely unmet need todevelop better ways of eliciting strong CD8+ T cells to protect againstinfection.

CD8+ T cells can recognize cancer antigens and cure malignancy: Similarto the situation with infectious agents, CD8+ T cells can also begenerated against tumor cell antigens. As exemplified byTumor-Infiltrating Lymphocytes (TILs), the passive administration ofanti-tumor CD8+ T cells can be sufficient to cure patients of advancedcancers in a small percentage of cases (Restifo et al., Nature ReviewsImmunology. 2012; 12(4):269-81). These CD8+ T cells recognize peptidestermed “tumor antigens” where the tumor contains antigens either notfound in normal tissue or present at much lower levels. As noted above,some tumor antigens are derived from tumorigenic viruses such as the E6and E7 antigens in HPV-related cervical cancer. Other tumor antigens arederived from mutations in germline proteins such as the V600E mutationin the BRAF protein. Yet other tumor antigens are normal proteins suchas HER-2/neu which is overexpressed in breast cancer, where the breastis a non-essential “disposable” tissue that can be sacrificed by animmune attack on breast-derived tissues. Here again, there is an urgentand largely unmet need to develop better ways of eliciting strong CD8+ Tcell responses to protect against cancer or treat patients with alreadyestablished malignant disease.

Numerous licensed vaccines are live, attenuated viruses (LAV): As notedabove, there is a major problem in the art which is that it has beendifficult to develop industrial applicable vaccines that are able togenerate antigen-specific CD8+ T cells. For viral infections, one of thebest ways is to generate anti-viral CD8+ T cells is to vaccinate with alive, attenuated virus (LAV) vaccine. Familiar examples of LAV vaccinesare the Measles/Mumps/Rubella (MMR) vaccine, Sabin poliovirus vaccine,FluMist® influenza vaccine, Yellow Fever Virus 17D vaccine, and Vacciniasmallpox vaccine. But it has been difficult to produce LAV vaccinesagainst viral infections for a variety of reasons that includeinefficient manufacturing process, a need for repeated vaccination withfollow-up “booster” vaccination many years later, and the generally poorquality and low level of the CD8+ T cell response to many vaccinecandidates.

CD8+ T cells can cure cancer in humans but are difficult to generate:For cancers not associated with viruses, there is no possibility ofdeveloping an LAV type vaccine. Instead, tumor antigens must beidentified or otherwise isolated or predicted and used for vaccination.To be curative for cancer, a substantial CD8+ T cell response is needed.This has been shown for regimens that isolate and expandtumor-infiltrating lymphocytes (TIL) which are CD8+ T cells grown exvivo and then administered back to the patients. In these studies, arelatively high number of TIL CD8+ T cells is required to successfullyeradicate and cure metastatic melanoma (Restifo et al., Nature ReviewsImmunology. 2012; 12(4):269-81). Many seemingly auspicious cancervaccines and immunotherapies turn out to be too weak to cure cancer whentested in vivo. For example, simply vaccinating with a tumor antigenpeptide emulsified in Montanide lipid as an immunostimulant fails tocure cancer because the resulting CD8+ T cells do not enter thecirculation and go to the tumors (Hailemichael et al., Nat Med. 2013;19(4):465-72. PMCID: 3618499).

CD8+ T cells are stimulated by antigen peptides presented on MHC Class I(MHC-I): In order to understand the process for generating CD8+ T cells,it is helpful to review how they arise during a normal immune response.CD8+ T cells are named because they have the CD8 protein on theirsurface. CD8 works as a “co-receptor” along with the T cell receptor(TCR) to recognize peptide antigens (typically 7-11 amino acids inlength) that are processed inside of cells by the cleavage of the intactproteins and then displayed on the surface of infected cells by majorhistocompatibility complex (MHC) Class I (MHC-I) molecules. These MHC-Imolecules hold the peptide antigen in a “groove” and the CD8+ T cellthen recognizes the peptide-MHC-I (pMHC-I) complex and becomesactivated. CD8+ T cells that kill the infected cell are termed“cytotoxic” but they can also interfere with infectious agents byproducing cytokines such as interferon-gamma (IFN-g).

Considering the foregoing, it is highly desirable to find anindustrially applicable means for producing vaccines that are highlyeffective for eliciting strong CD8+ T cells, CD4+ T cells, and antibodyresponses against infectious agents and tumor antigens.

Need for antigen-presenting cells (APC) to generate antigen-specificCD8+ T cells: With this as an introduction, it can be appreciated that akey event in the generation of CD8+ T cells is to develop a cell typecalled an “antigen-presenting cell” (APC) that can present pMHC-I touneducated or naive CD8+ T cell precursors to induce them to divide,expand in numbers, and persist for prolonged periods as highly active“memory” CD8+ T cells. To be effective at generating CD8+ T cells, anAPC must both express peptide antigen on MHC-I (pMHC-I) that isrecognized by the TCR (called “Signal 1”) and also co-stimulate theresponding cells through additional receptor (called “Signal 2”) andeven other receptors (called “Signal 3”). TCR stimulation by pMHC-Iprovides Signal 1 and generally stimulation of the CD28 receptor on CD8+T cells provides Signal 2. Signal 3 can be provided in a non-redundantfashion either by soluble proteins such as interferon-alpha (Type Iinterferon) and/or interleukin-12 (IL-12) and/or cell surface moleculessuch as CD27 ligand (CD27L, also called CD70 or TNFSF7), 4-1BBL (alsocalled CD137L or TNFSF9), and/or OX40L (also called CD134L or TNFSF4)(Sanchez and Kedl, Vaccine. 2012; 30(6):1154-61. PMCID: 3269501). Whatis needed is a vaccine approach that can activate an APC to provide allof these signals. This requires a good dendritic cell stimulus, alsocalled an “immune adjuvant” or “adjuvant.”

APC cross-presentation of extracellular antigens: The first requirementfor an APC is to express peptide antigen on MHC-I (pMHC-I). Theprototypic APC is the dendritic cell which takes up protein antigensfrom its environment, degrades these proteins into peptides, loads theresulting peptides onto MHC-I, and then presents the pMHC-I on theirsurface to provide the TCR stimulus that is Signal 1. This process isvery different from cells infected by a microbial pathogen or tumorcells. In those cases, the protein antigen is produced within the cellitself—not taken up from the extracellular space—and then proteindegradation products (which are peptides) are loaded onto MHC-I andexported to the cell surface as pMHC-I to provide Signal 1. What makesdendritic cells and other APCs special is that they can form pMHC-I fromproteins in their environment, a phenomenon termed “cross-presentation.”For dendritic cells to do this, they must take up the protein antigenfrom their environment using one of a few very specialized receptors,including DEC205, CD11c, BDCA1, BDCA3, and/or CD40. After taking upprotein antigen from the extracellular space, these receptors direct thedelivery of the protein antigen into membrane-limited intracellularcompartments (“endosomes”) where the protein can be digested intopeptides and then transferred into compartments where MHC-I is beingassembled. Of special important to the instant invention is that thebest receptor on dendritic cells for processing protein antigen intopMHC-I (i.e., crosspresentation) is the CD40 receptor (Chatterjee etal., Blood. 2012; 120(10):2011-20; Cohn et al., J Exp Med. 2013;210(5):1049-63. PMCID: 3646496). Therefore, it is highly desirable for avaccine to include a protein antigen that is targeted toward the CD40receptor on dendritic cells.

Activation of the APC stimulates crosspresentation: A second requirementfor an APC to crosspresent an exogenous protein antigen is for the APCto be “activated.” For dendritic cells, such activation is ideallyprovided by an effective stimulus through the CD40 receptor, whichpromotes crosspresentation and the formation of the pMHC-I Signal 1(Delamarre et al., J Exp Med. 2003; 198(1):111-22). Similarly, B cells,which are another type of APC, can be activated by a CD40 receptorstimulus to crosspresent soluble protein antigens (Ahmadi et al.,Immunology. 2008; 124(1):129-40).

Crosspresentation of antigen by dendritic cells in the absence of CD40stimulation leads to CD8+ T cell tolerance: DEC-205 is a receptor ondendritic cells and B cells recognized on mouse cells by the NLDC-145monoclonal antibody (Inaba et al., Cellular immunology. 1995;163(1):148-56). Bonifaz et al. showed that the binding portion of ananti-DEC205 antibody can be genetically fused to a model antigen,chicken ovalbumin (OVA). The injection of anti-DEC205/OVA fusion proteindirects the OVA antigen to dendritic cells and leads tocrosspresentation of OVA peptide antigen on MHC-I. However, while thistreatment induces anti-OVA CD8+ T cells to divide and proliferate, thesecells soon die off and are deleted. This results in specific tolerancefor OVA that cannot be overcome by subsequent vaccination with OVA plusComplete Freund's Adjuvant (CFA), which is usually considered to be agold standard for vaccination (although CFA is far too inflammatory tobe used in humans). However, if anti-DEC205/OVA fusion protein iscombined with a stimulus for the CD40 receptor, then very stronganti-OVA CD8+ T cell responses result (Bonifaz et al., J Exp Med. 2002;196(12):1627-38. PMCID: 2196060). This indicates that simply targetingantigens to dendritic cells alone (e.g., using a fusion protein ofanti-DEC205 and antigen) does not succeed in eliciting high levels ofefficacious and persisting antigen-specific CD8+ T cells. In fact, itshows that allowing antigen to be taken up by unactivated dendriticcells should be avoided because it will work against the goal ofcreating strong antigen-specific CD8+ T cell responses.

Generating CD8+ T cell responses is best when antigen is delivered todendritic cells in conjunction with an adjuvant: Although they did notuse a CD40 stimulus, Kamath et al. (J Immunol. 2012; 188(10):4828-37)developed a vaccine system for delivering an antigen either directlyattached to an antigen or co-delivered with a separate, unattachedantigen. When antigen was delivered to DCs in the absence of adjuvant,antigen-specific T cells were induced to proliferate but did notsubsequently differentiate into effector cells. Instead, effectiveimmunity was only induced when the test vaccine provided antigen andadjuvant to the same individual DCs within a short window of time. Theseparameters are fulfilled when the antigen and adjuvant are linked intime and space as parts of the very same molecule, as provided by theinstant invention.

To fulfill the need for a vaccine that induces a strong CD8+ T cellresponses, the instant invention provides for a composition thatcontains, for example, CD40 ligand (CD40L, TNFSF5, which is an agonistof the CD40 receptor) physically linked to a multimerization domain thatorganizes it into a highly active many-trimer structure in addition tobeing physically linked to an antigen. In this way, antigen can betargeted to dendritic cells via binding to the CD40 receptor on theirsurface and activates the dendritic cell simultaneously. Thisarrangement can thus avoid delivery of antigen to dendritic cells thatdo not become activated and which instead would induce antigen-specificCD8+ T cell tolerance. As a result, the compositions of the instantinvention provide for a unusually high level of activity in inducingstrong CD8+ T cell responses, where the TCRs of elicited CD8+ T cellsshow an exceptionally high level of avidity for pMHC-I and where avaccine of the invention confers surprisingly profound protection fromchallenge by an infectious agent (Vaccinia encoding HIV-1 Gag as a modelantigen). Variations on these compositions are expected to elicit verystrong CD4+ T cells and B cell antibody responses in a similar fashion.

SUMMARY OF THE INVENTION

The invention provides fusion proteins comprising antigens of infectiousdisease agents and cancer cells linked to many-trimer forms of TNFSuperFamily (TNFSF) ligands. The TNFSFs serve as vaccine adjuvants forincreasing the immune response to the antigens. In particular, a fusionpolypeptide strand that self-assembles inside cells into a many-trimerform of CD40 ligand (CD40L, TNFSF5) was shown to elicit surprisinglystrong responses against an infectious disease agent and a tumorantigen. Other similar fusion proteins are contemplated and theirconstruction provided for in the application. The fusion proteins can bedelivered to a host either as nucleic acids used directly in DNAvaccination or carried and expressed by a viral vector such asadenovirus. It is contemplated that isolated fusion proteins could bealso be administered with good effect. In addition to use as a vaccineto prevent or ameliorate disease caused by an infectious agent,compositions of the invention may be used for the treatment of ongoinginfection or for cancer immunotherapy.

To create a vaccine that effectively elicits strong CD8+ T cellresponses, highly active forms of TNF Superfamily ligands (TNFSFs) wereconstructed as fusion proteins with test antigens from infectiousdisease agents and tumors. Using CD40 ligand (CD40L, also called TNFSF5)as an exemplary TNFSF, the resulting fusion proteins were given to micein the form of a DNA vaccine (by injection of plasmid DNA into muscle)as a means to deliver antigen to dendritic cells and activate thesecells through their CD40 receptor at the same time. This approachminimizes the separate delivery of antigen to dendritic cells that havenot been activated by adjuvant, which would otherwise result in CD8+ Tcell tolerance as shown by Bonifaz et al. (J Exp Med. 2002;196(12):1627-38. PMCID: 2196060) and Kamath et al. (J Immunol. 2012;188(10):4828-37). In the exemplary case, this invention combines one ofthe best vaccine adjuvants for dendritic cell activation (i.e., CD40L)along with targeting the antigen to dendritic cells by virtue of theantigen being operatively linked to CD40L (the ligand for the CD40receptor) which binds to CD40 and delivers the antigen to dendriticcells for cross-presentation as pMHC-I. Previous attempts to link CD40Lwith antigen were flawed by defective molecular design and did notresult in such a powerful vaccine. Instead, the approach of the instantinvention provides a combination in such a way as to provide asurprisingly strong CD8+ T cell response that is highly protective. Byselecting the appropriate antigen(s) and TNFSFs and an appropriatedelivery method, applications include vaccines against infectious agentsand malignant cells. Using fusion proteins directly or as their encodingnucleic acid sequences delivered by a DNA or RNA vaccine or by a viralvector such as adenovirus, the invention has substantial industrialapplication.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic drawing illustrating the need to cluster a TNFSFreceptor such as the CD40 receptor on dendritic cells and other APCs inorder to provide a strong cell stimulus. This requirement for clusteringaffects the design of an effective form of TNFSF ligand or an anti-TNFSFreceptor-binding antibody.

FIG. 2: Agonistic anti-CD40 antibodies can cluster CD40 receptors solong as they bind to and are “mounted” on a nearby cell that expressesreceptors for the Fc tail of the antibody molecule. Abbreviations:FcγR—the receptor for the Fc portion of immunoglobulin G (IgG).Anti-CD40 MAb—a monoclonal antibody that binds to CD40.

FIG. 3: Molecular design of fusion proteins that create many-trimerforms of soluble CD40L. On the left is a schematic for a 1-trimer formof CD40L that cannot cluster the CD40 receptor and as a result isinactive, as shown by Haswell et al. (Eur J Immunol. 2001;31(10):3094-100) and Holler et al. (Mol Cell Biol. 2003; 23(4):1428-40)and described in EP 1246925 B1. As previously described (Stone et al., JVirol. 2006; 80(4):1762-72) and presented in U.S. Pat. No. 7,300,774 B1and U.S. Pat. No. 7,332,298 B2, and also in EP 1246925 B1, theextracellular domain (ECD) of CD40L can be genetically fused toscaffold-forming proteins such as Acrp30 (middle) or surfactant proteinD (SPD) (right). The 2-trimer Acrp30-CD40L protein is also calledMegaCD40L™ or CD40L hexamer, whereas the 4-trimer SP-D-CD40L protein isalso called UltraCD40L™. These many-trimer forms of CD40L can clusterthe CD40 receptor and act as a vaccine adjuvant. This occurs in part byactivating dendritic cells (Miconnet and Pantaleo, Vaccine. 2008;26(32):4006-14).

FIG. 4: Two- and four-trimer CD40L fusion proteins are vaccine adjuvantsfor CD8+ T cell responses. Mice were vaccinated by injecting “naked”plasmid DNA into muscle in order to test different forms of CD40L as anadjuvant for the HIV-1 Gag antigen. In Panel A, CD8+ T cell responseswere detected as killing of P815 target cells pulsed with Gag peptide.In Panel B, CD8+ T cell responses were detected by measuring the numberof individual interferon-gamma secreting cells in response to Gagpeptide antigen using an ELISPOT assay. There was a distinct improvementin CD8+ T cell responses using a 2-trimer form of CD40L (Acrp30-CD40L)and more preferably a 4-trimer form of CD40L (SPD-CD40L) (Stone et al.,J Virol. 2006; 80(4):1762-72). To show the general applicability of thisapproach, a similar vaccine assay system was used to show that otherTNFSF ligands could be multimerized as 4-trimer proteins and used asvaccine adjuvants, including GITRL, 4-1BBL, OX40L, RANKL, LIGHT, CD70,and BAFF (Kanagavelu et al., Vaccine. 2012; 30(4):691-702. PMCID:3253891).

FIG. 5: Molecular design of multimeric CD40L fusion proteins containingan in-frame insertion encoding HIV-1 Gag as a model antigen. Top:pSPD-Gag-CD40L is a plasmid containing an antigen inserted into theprotein strand that results in a 4-trimer form of CD40L. At the nucleicacid level, the codons for a model antigen, HIV-1 Gag, were positionedinto the coding sequence of the SPD-CD40L construct. In the resultingtranslated protein, the N-terminus is comprised of a secretion signalpeptide from SPD followed by an N-terminal sequence of SPD termed the“hub” which contains 2 cysteines in each strand, thereby producingdisulfide bonds that (a) covalently couple three individual polypeptidestrands together to form an “arm” and (b) covalently couple 4 trimericarms into the final 12-chain, 4-arm structure shown in the bottom leftof the figure (where the inserted Gag antigen is shown as a solid bulgein each arm of the protein). Note that the Gag antigen sequence waspositioned between the 105 and 106 amino acids of murine SPD protein,while retaining the previously constructed CD40L domain at theC-terminal end. Like the parent SPD-CD40L molecule, this protein strandof SPD-Gag-CD40L spontaneously self-assembles inside cells into amultimeric, many-trimer form of CD40L that is then secreted into theextracellular space. 2nd from Top: pTrimer-Gag-CD40L (labeledpTr-Gag-CD40L) is a plasmid constructed by deleting codons for aminoacids 24-105 of murine SPD. This removes the hub region containing the2-cysteines. Also included is the t-PA signal peptide sequence forsecretion. This results in the production of a single-trimer, 1 “arm”form of the Gag antigen-CD40L protein, as shown in the bottom right ofthe figure (where the Gag antigen is shown as a solid bulge in this1-trimer form of CD40L). 3rd from Top: pGag is the plasmid encodingamino acids for the p55 Gag antigen preceded by the t-PA signal sequencefor secretion, as described by Qiu et al. (J Virol. 1999;73(11):9145-52). This is a control antigen construct that has no CD40Ladjuvant. 4th from Top: pSPD-CD40L is the plasmid encoding a 4-trimerform of CD40L previously described by Stone et al. (J Virol. 2006;80(4):1762-72) and in U.S. Pat. No. 7,300,774 B1 and U.S. Pat. No.7,332,298 B2. This is an adjuvant-only protein that does not contain anantigen. It can, however, be co-administered with an antigen plasmidsuch as pGag, as shown in FIG. 4.

FIG. 6: pSPD-Gag-CD40L encodes a secreted protein. Panel A shows aWestern blot of a reducing SDS-PAGE gel analysis of the culture media of293T cells were transiently transfected with DNA for the plasmids shown.An antibody for murine CD40L was used to reveal the protein bands. Asshown, pSPD-Gag-CD40L encodes a single protein of the expected size of105 kDa. A single 105 kDa band was also observed using antibody to thep24 portion of Gag (not shown). Panel B shows a similar analysis usingnon-denaturing PAGE in the absence of a reducing agent. Multiple bandswere observed at >200 kDa molecular weight, demonstrating the formationof large multimeric complexes. As is commonly observed in such analysesof collagen-like proteins, partial denaturation during processing canresult in an unwinding of some of the collagen triple helix, which couldthus lead to a less compact protein that moves more slowly through thegel during the electrophoretic process.

FIG. 7: Qualifying assay for the biological activity of SPD-Gag-CD40L invitro. Panel A: In vitro activity using a CD40 receptor NF-κB indicatorcell line. To produce soluble protein, 293T cells were transientlytransfected with plasmids for pcDNA3.1 (empty vector control),pSPD-CD40L, or pSPD-Gag-CD40L and the protein-containing supernatantswere collected 48 hours later. To determine the activity of the CD40L inthese proteins, the culture media as added to cultures of 293 reportercells containing an NF-κB-driven gene for secreted alkaline phosphatase(SEAP) and expressing the CD40 receptor (CD40-293-SEAP reporter cells).If the CD40 receptor is activated by CD40L, then NF-κB-driven SEAPproduction results in the secretion of SEAP which can be measured by acolorimetric enzyme assay at OD650 (Maurais et al., Virology. 2009;385(1):227-32). In this assay, a single trimer of CD40L (R&D Systems,Inc., Minneapolis, Minn.) was entirely inactive and did not induce SEAPproduction (not shown), indicating the strict requirement for amany-trimer form of CD40L for activity in this assay. In contrast, boththe pSPD-CD40L adjuvant protein and the new SPD-Gag-CD40L protein of theinstant invention were active as CD40 receptor activators. Panel B:Stimulating activity on mouse bone marrow-derived dendritic cells(BMDDC). As in Panel A, culture supernatants from 293T cells transfectedwith pcDNA3.1 or pSPD-Gag-CD40L were incubated with BMDDC for 18 hours.Cells were washed, stained with fluorochrome-conjugated antibodies, andassayed by flow cytometry for the expression of activation andmaturation markers. The SPD-Gag-CD40L protein upregulated CD80 andespecially CD86 and CCR7, indicating that this fusion protein was fullycapable of activating normal dendritic cells. As expected, the CD40receptor was downregulated by exposure to SPD-Gag-CD40L. A cytokine mixwas used as a positive control (“Mimic,” consisting of 10 ng/ml ofrhTNF-alpha, 10 ng/ml of rhIL-1beta, 1000 U/ml of rhIL-6 and 1 μg/ml ofPGE2; Sato et al., Cancer Sci. 2003; 94(12):1091-8). * p<0.05, **p<0.01, and *** p<0.001 compared to pcDNA3.1 supernatant. Datarepresents independent wells in the same experiment.

FIG. 8: DNA vaccination with pSPD-Gag-CD40L demonstrates a surprisinglyhigh level of CD8+ T cell responses. Panel A: DNA vaccination schedule.Mice were vaccinated three times at two-week intervals with anintramuscular injection of 100 μg of plasmid DNAs. Panels B and C: CD8+ELISPOT assay. To measure the Gag-specific CD8+ T cell response, spleencells were collected 14 days after the last vaccination and tested byELISPOT assays. Panel B shows cells producing interferon-gamma and PanelC shows cells producing IL-2. The control vaccination is pGag+pcDNAwhere empty pcDNA3.1 (pcDNA) was used to keep the total amount of DNAconstant. The previously reported mix of antigen and 4-trimer CD40Ladjuvant plasmid is pGag+pSPD-CD40L which consists of separate plasmidsfor antigen and adjuvant, i.e., not present in the same secretedmolecule. Surprisingly, pSPD-Gag-CD40L, the subject of the instantinvention, resulted in a massive antigen-specific CD8+ T cell response(note that a broken Y-axis is needed to keep the results visible in thegraph). In contrast, pGag+pIL-12 gave more modest CD8+ T cell responses,even though a pIL-12 plasmid is currently being evaluated in humanvaccine trials. Panel C shows the same analysis using IL-2 ELISPOT assayand showed the surprising strength of pSPD-Gag-CD40L, the subject of theinstant invention.

FIG. 9: DNA vaccination with pSPD-Gag-CD40L demonstrates a surprisingimprovement in CD8+ T cell quality. Panel A: T cell receptor avidity forpeptide antigen/MHC-I measured by ELISPOT assay. Splenocytes werecultured with serial dilutions of CD8+ T cell specific peptide AMQMLKETIfor 18 hours. Splenocytes from mice vaccinated with pSPD-Gag-CD40Linduced a significant increase in IFN-γ ELISPOTs following stimulationwith Gag peptide AMQMLKETI at a concentration of 1 ng/ml and 10 pg/mlwhereas there was essentially no activity at these doses usingsplenocytes from mice vaccinated with pGag antigen alone or a mixture ofseparate plasmids for pGag and pSPD-CD40L adjuvant. * p<0.05; ** p<0.01;*** p<0.001 compared to pGag alone or pGag+SPD-CD40L vaccination. PanelB: IgG antibody responses against Gag antigen. Total IgG specific forGag was measured by ELISA assay from mouse serum collected on day 42.Consistent with a previous study (Stone et al., J Virol. 2006;80(4):1762-72), CD40L adjuvant used in this format is not an adjuvantfor antibody responses.

FIG. 10: The multi-trimer structure of SPD-Gag-CD40L is necessary forthe improved vaccine effect. In Panels A and B, pTrimer-Gag-CD40L wasused as 1-trimer control for 4-trimer pSPD-Gag-CD40L. As shown, themany-trimer structure was necessary for the strong adjuvant effect.

FIG. 11: Protective effects of pSPD-Gag-CD40L vaccination measured byvaccinia-Gag viral challenge. BALB/c female mice were immunizedintramuscularly with the plasmids shown on days 0, 14, and 28. Two weeksfollowing the final vaccination, the mice were challengedintraperitoneally with 10E7 plaque-forming units (PFU) of vaccinia-Gag.Mice were sacrificed 5 days after viral challenge and the ovaries wereharvested and analyzed for PFU. Panel A: Intramuscular DNA vaccinationwith pSPD-Gag-CD40L resulted in significantly greater protection fromviral challenge. In contrast, DNA vaccination with a mixture of pGagantigen plus pSPD-CD40L adjuvant as separate plasmids only induced amodest reduction in viral loads that was not significantly reducedcompared to pGag antigen alone. * p<0.05; ** p<0.01; *** p<0.001. PanelB: Evaluation of a single trimer pTrimer-Gag-CD40L construct. As shownbefore, the multi-trimer structure of SPD-Gag-CD40L is necessary for theimproved vaccine effect.

FIG. 12: Adenoviral vector delivery of SPD-Gag-CD40L is surprisinglyprotective against virus challenge. BALB/c female mice were immunizedintramuscularly on days 0 and 14 with adenovirus 5 (Ads) expressing theHIV-1 Gag antigen (Ads-Gag) or the SPD-Gag-CD40L construct(Ad5-SPD-Gag-CD40L). Two weeks following the final vaccination, micewere challenged intraperitoneally with vaccinia-Gag virus (10E7 PFU).Mice were sacrificed 5 days later and ovaries were harvested forvaccinia PFU determinations. Surprisingly, Ad5-SPD-Gag-CD40L vaccinationreduced viral load by ˜7 logs following vaccinia-Gag challenge. Nodetectable virus could be found in the mice that had received thisvaccine, indicating complete protection (sterilizing immunity).

FIG. 13: Construction and Western blot of SPD-gp100-CD40L. Panel A:Model of SPD-gp100-CD40L fusion. Amino acids 25 to 596 (sequence KVPRNQDto EAGLGQV) of human gp100 was inserted between amino acids 105 and 106of murine SPD within the SPD-CD40L fusion construct. Panel B: Schematicdiagram of expected SPD-gp100-CD40L 4-trimer structure. Panel C: Westernblot analysis. 293T cells were transfected with DNA plasmid encodinggp100 or the SPD-gp100-CD40L fusion protein. After 48-hour culture,supernatant was collected and run on an SDS-PAGE gel in the presence ofreducing agent. Western blot was performed using a polyclonal antibodyto gp100.

FIG. 14: Biological activity of SPD-gp100-CD40L. Panel A: In vitroactivity of SPD-CD40L and SPD-gp100-CD40L was determined using acell-based CD40 NF-kB enzymatic reporter system. An equivalent amount of293T supernatant from pcDNA3.1, pSPD-CD40L or pSPD-gp100-CD40Ltransfected cells was incubated with 293-CD40-SEAP NF-kB reporter cells.Panel B: In vitro activity of SPD-gp100-CD40L was evaluated on mousebone marrow derived mouse DC and compared to empty vector or Mimiccytokine positive control. * p<0.05, ** p<0.01 by Student's t testcompared to pcDNA3.1 supernatant.

FIG. 15: Immunotherapy of established B16F10 melanoma tumors. Panel A:Immunization schedule for B16-F10 tumor challenge and DNA/GVAXtherapeutic vaccination, as indicated by arrows. B16F10 cells (50,000)were injected i.d. into the left flank of C57BL/6 mice on day 0. Micewere then immunized by i.m. injection of PBS or pSPD-gp100-CD40L plasmidon day 3, 10, and 17. GVAX, B16F10 tumor cells expressing GM-CSF, wereirradiated at 5,000 rad and 1×10E6 cells injected subcutaneously on day3, 6, and 9. Panel B: Tumor growth analysis. Each point represents themean tumor volume in each group (n=5). We did not observe a statisticaldifference in tumor sizes between no treatment (PBS) and SPD-gp100-CD40Lvaccination groups. Panel C: Survival analysis based on the date ofdeath or when tumor size reached >1500 cm2. No statistical differencesin survival were observed between groups.

FIG. 16: Immunotherapy of established B16F10 melanoma tumors by DNAvaccination with a combination of pSPD-gp100-CD40L, pIL-12p70 andpGM-CSF. Panel A: Immunization schedule for B16F10 tumor challenge andDNA/GVAX vaccination, as indicated by arrows. B16F10 cells (50,000) wereinjected i.d. into the left flank of the mice on day 0. Mice wereimmunized i.m. with PBS, pSPD-gp100-CD40L+pIL-12,pSPD-gp100-CD40L+pGM-CSF, or pSPD-gp100-CD40L+pIL-12+pGM-CSF on day 3,10, and 17. For GVAX therapy B16-F10 tumor cells expressing GM-CSF(GVAX), were irradiated at 5,000 rad and 1×106 cells were injectedsubcutaneously on day 3, 6, and 9. Panel B: Tumor growth analysis. Eachpoint represents the mean tumor volume of animals in each group (n=5).There was a significant reduction in tumor growth kinetics forSPD-gp100-CD40L+IL-12+GM-CSF vaccinated mice compared to other groups.(** p<0.01; *** p<0.001 compared to PBS or SPD-gp100-CD40L+IL-12 orSPD-gp100-CD40L+GM-CSF vaccination groups). Panel C: Survival analysisof mice. We observed a significant increase in survival and tumor freesurvival (date of tumor appearance) for pSPD-gp100-CD40L+pIL-12+pGM-CSFvaccinated mice as compared to other groups (** p<0.01; *** p<0.001compared to PBS, pSPD-gp100-CD40L+pIL-12, or pSPD-gp100-CD40L pGM-CSFvaccination groups). Panel D: Tumor growth kinetics of individual micefrom each treatment group.

FIG. 17: Separate expression of gp100 and SPD-CD40L proteins fails toinduce anti-tumor activity. As a control for pSPD-gp100-CD40L, severalother anti-tumor treatment approaches were tested and found to beinferior. Panel A: Immunization schedule for B16F10 tumor challenge andDNA vaccination, as indicated by arrows. B16-F10 cells (50,000) wereinjected into the left flank of the mice on day 0. Mice were immunizedi.m. with PBS, pgp100, pgp100+pIL-12, pgp100+pGM-CSF,pgp100+pIL-12+pGM-CSF, or pgp100-IRES-SPD-CD40L+pIL-12+pGM-CSF on day 3,10, and 17. Panel B: Tumor growth analysis. Each point represents themean tumor volume of animals in each group (n=5). We did not observe anystatistical difference in tumor size between vaccination groups. PanelC: Survival analysis. We did not observe any statistical difference insurvival of mice between groups.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO 1: DNA sequence for muSP-D-Gag-muSP-D-muCD40L. This is the DNAsequence of a fusion protein using the murine sequences for SPD andCD40L. Due to minor differences between species, it is preferable to usea murine sequence for administration to mice, a macaque sequence foradministration in monkeys (Stone et al., Clin Vaccine Immunol. 2006;13(11):1223-30), a human sequence for administration to humans, and so.This minimizes the possibility of antibodies forming against axenogeneic protein, other than the antigen contained in the construct.In this example, what is shown is the nucleic acid sequence used for theexperiments shown in FIGS. 6-12. (Note that surfactant protein D isvariously abbreviated as either ‘SPD’ or ‘SP-D’. The location of the Gagantigen insert is shown in non-italicized type face.

SEQ ID NO 2: Protein sequence for muSP-D-Gag-muSP-D-muCD40L. This is thetranslation of SEQ ID NO 1.

SEQ ID NO 3: DNA sequence for tpa-muACRP30-gp120-muACRP30-muBAFF. Thisis a DNA sequence of a fusion protein using the previously described2-trimer form of Acrp30-BAFF into which has been inserted a DNA sequenceof HIV-1 gp120 envelope as an antigen. It is contemplated that the2-trimer fusion protein encoded by this nucleic acid sequence willactivate the Env gp120-binding B cell receptor (BCR) on B cells andsimultaneously engage receptors for BAFF on these B cells that synergizewith BCR engagement to stimulate the B cell to produce anti-Envantibodies.

SEQ ID NO 4: Protein sequence for tpa-muACRP30-gp120-muACRP30-muBAFF.This is the translation of SEQ ID NO 3.

SEQ ID NO 5: DNA sequence for muSP-D 100-muSP-D-muCD40L. This is the DNAsequence of a fusion protein using the murine sequences for SPD andCD40L. The inserted antigen (non-italicized sequence) is encoded by thenucleotide sequence for human gp100, a xenogenic antigen that has beenfound to be useful in melanoma studies in mice (Gold et al., J Immunol.2003; 170(10):5188-94).

SEQ ID NO 6: Protein sequence for muSP-D-gp100-muSP-D-muCD40L. This isthe translation of SEQ ID NO 5.

SEQ ID NO 7: DNA sequence for tpa-huIgG1Fc-gp120-GCN4-huAPRIL. This is aDNA sequence encoding a human t-PA signal sequence for protein secretionjoined in-frame with the human IgG1 Fc region joined in-frame with HIV-1Env gp120 joined in-frame with the GCN4 trimerization motif joinedin-frame with the extracellular domain of human APRIL. It iscontemplated that the 2-trimer fusion protein encoded by this nucleicacid sequence will activate the Env gp120-binding B cell receptor (BCR)on B cells and simultaneously engage receptors for APRIL on these Bcells that synergize with BCR engagement to stimulate the B cell toproduce anti-Env antibodies.

SEQ ID NO 8: Protein sequence for tpa-huIgG1Fc-gp120-GCN4-huAPRIL. Thisis the translation of SEQ ID NO 7.

SEQ ID NO 9: DNA sequence for huSP-D-NP-huSP-D-huCD40L-NST. It waspreviously found that some embodiments of SPD-CD40L can be equally ormore active when the extracellular “stalk” region of CD40L is deleted.This stalk links the CD40L trimeric extracellular domain (ECD) with thetransmembrane region that holds CD40L in the membrane. The SPD-CD40L-NSTconstruct is disclosed in US 2009/0081157 A1 (see especially FIG. 21,Examples 1, 11, and 13) which is incorporated by reference. The instantsequence comprises an insertion of coding sequences for thenucleoprotein (NP) antigen from influenza A. It is contemplated that the4-trimer fusion protein encoded by this nucleic acid sequence willelicit strong CD8+ T responses against this conserved influenza antigen.

SEQ ID NO 10: Protein sequence for huSP-D-NP-huSP-D-huCD40L-NST. This isthe translation of SEQ ID NO 9.

SEQ ID NO 11: DNA sequence for tpa-muACRP3O-CSP1-muACRP30-muCD40L. Thisis a DNA sequence encoding a human t-PA signal sequence for proteinsecretion joined in-frame with a portion of the murine Acrp30 sequencejoined in-frame with codons for the circumsporozoite protein-1 (CSP-1)of Plasmodium yoelii joined in-frame with a portion of the murine Acrp30sequence joined in-frame with the extracellular domain of murine CD40L.Plasmodium yoelii is used for malaria vaccine studies because it causesa malaria-like disease in mice. CD8+ T cells directed against the CSP-1antigen of this agent can provide immunity to malaria (Sedegah et at,Proc Natl Acad Sci USA. 1998; 95(13):7648-53). It is contemplated thatmice vaccinated with this construct will be resistant to disease causedby intravenous challenge with Plasmodium yoelii-infected red bloodcells.

SEQ ID NO 12: Protein sequence for tpa-muACRP3O-CSP1-muACRP30-muCD40L.This is the translation of SEQ ID NO 11.

SEQ ID NO 13: DNA sequence for muSP-D-Gag-muSP-D-muRANKL. This is a DNAsequence encoding a portion of the murine SPD sequence joined in-framewith codons for HIV-1 Gag antigen joined in-frame with a portion of themurine Acrp30 sequence joined in-frame with the extracellular domain ofmurine RANKL. Of special note is the difference of position in placingthe antigen within the sequence of the SPD “arms,” in this case shiftedtoward the 5′ end (or N-terminal end in the protein) the equivalent of10 codons in the SPD sequence. It is contemplated that this constructused as a vaccine will elicit strong immune responses in mice.

SEQ ID NO 14: Protein sequence for muSP-D-Gag-muSP-D-muRANKL. This isthe translation of SEQ ID NO 13.

SEQ ID NO 15: DNA sequence of huSP-D-WT1-huSP-D-huCD40L. This is a DNAsequence encoding a portion of the human SPD sequence joined in-framewith codons for the human WT1 protein joined in-frame with a portion ofthe human SPD sequence joined in-frame with the extracellular domain ofhuman CD40L. WT1 is a tumor antigen present in many types of humancancer (Chaise et al., Blood. 2008; 112(7):2956-64). It is contemplatedthat this construct used as a vaccine will elicit strong immuneresponses in humans against cancer cells expressing the WT1 tumorantigen.

SEQ ID NO 16: Protein sequence for huSP-D-WT1-huSP-D-huCD40L. This isthe translation of SEQ ID NO 15.

SEQ ID NO 17: DNA sequence of muSP-D-MAGE-A3-muSP-D-muBAFF. This is acontemplated DNA sequence encoding a portion of the murine SPD sequencejoined in-frame with codons for the human MAGE-A3 tumor antigen (Groeperet al., Int J Cancer. 2007; 120(2):337-43) joined in-frame with aportion of the murine SPD sequence joined in-frame with theextracellular domain of murine BAFF. Of note is that codons for 20 aminoacids (PPGLPGIPGPMGARASVLSG) in the N-terminal half of the SPD arm havebeen deleted. This exemplifies how the SPD “arms” can be shortenedN-terminal to the insertion site of the antigen sequence. Similardeletions in the C-terminal half of the SPD arm are also contemplated,as are deletions in both sides of the SPD arms that flank the antigensequence insertion site.

SEQ ID NO 18: Protein sequence of muSP-D-MAGE-A3-muSP-D-muBAFF. This isthe translation of SEQ ID NO 17.

DEFINITIONS

This disclosure uses art-recognized concepts and methods. The skilledartisan will be familiar with resources including the following:“Janeway's Immunology” by Kenneth Murphy, Garland Science Press, 2011;“Fundamental Immunology” by William E. Paul, Lippincott Williams &Wilkins, 2008; “Cellular and Molecular Immunology, 7th Edition” by AbulK. Abbas, Andrew H. H. Lichtman, and Shiv Pillai, Elsevier Press, 2011;“Current Protocols in Immunology,” Wiley Press, 2012; and “CurrentProtocols in Molecular Biology,” Wiley Press, 2012. In addition, thefollowing patents and applications are incorporated by reference: U.S.Pat. No. 7,300,774B1; U.S. Pat. No. 7,332,298 B2; US 2009/0081157 A1.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology can be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCR Publishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. It is further to be understood that all base sizes or aminoacid sizes, and all molecular weight or molecular mass values, given fornucleic acids or polypeptides are approximate, and are provided fordescription. Although methods and materials similar or equivalent tothose described herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. The term“comprises” means “includes.” The abbreviation, “e.g.” is derived fromthe Latin exempli gratia, and is used herein to indicate a non-limitingexample. Thus, the abbreviation “e.g.” is synonymous with the term “forexample.”

“C1q family protein” refers to a member of the C1q family. Exemplary C1qfamily proteins include, but are not limited to, C11, Acrp30, and HIB27.Preference is given to Acrp30. Like the collectins, C1q family membershave 2 or more trimeric, collagen-like “arms” that provide themultivalent structures of these molecules. The instant inventionutilized C1q family proteins as a multimerization scaffold by replacingtheir normal C-terminal “C1q” domains with a TNFSF receptor binding suchas the ECD of a TNFSF ligand.

“Collectin” refers to a member of the collectin family. See URLhttp://en.wikipedia.org/wiki/Collectin for a listing of collectins andtheir gene names. They include pulmonary surfactant A, pulmonarysurfactant D, conglutinin, collectin-43, mannose-binding protein MBL1 orMBL2, and others. Preference is given to surfactant protein D(abbreviated alternatively as SP-D or SPD). All collectins have two ormore trimeric collagen-like “arms” joined in the center at a “hub” andradiating outward to display their C-terminal ends. Each collectin has aC-terminal domain that typically binds to carbohydrate. When used as amultimerization scaffold in the instant invention, each collectin ismade without the natural C-terminal end and a TNFSF ECD receptor bindingdomain is placed there instead. Preference is given to surfactantprotein D which has four trimeric arms ending C-terminally.

“Complete TNFSF receptor” is a term used herein in marked distinction toa single polypeptide chain often referred to as a TNFSF receptor protein(see URL http://www.genenames.org/genefamilies/TNFRSF for a listing ofTNFSF receptors (also called TNFRSFs) and their gene names. Thenucleotide and peptide sequences of single TNFSF receptor polypeptidechains are listed in GenBank, SwissProt, and other databases. However,in actuality, single TNFSF receptor polypeptide chains are not found inisolation on the surface of cells. Instead, two or more TNFSF receptorchains are co-localized or linked As an example, the Fas receptor (CD95)for Fas ligand (FasL) is held together in the absence of FasL by theirN-terminal “pre-ligand association domains” or PLAD (Siegel et al.,Science. 2000; 288(5475):2351-4). Similarly, there is a domain in theextracellular region of CD40 that holds this receptor together as 2 ormore chains (Smulski et al., J Biol Chem. 2013). Consequently,stimulation of TNFSF receptors generally does not involve simplebringing together of 2 or more receptor chains. When the ligand doesbind to the receptor, computer modeling suggests that a ligand trimerengages three receptor chains (Bajorath et al., Biochemistry. 1995;34(31):9884-92). Thus, this application uses the term “complete TNFSFreceptor” to indicate that binding to a TNFSF receptor involves bindingto 2 or preferably 3 receptor protein chains.

“Immune system” refers to T cells, B cells, NK cells, dendritic cells,monocytes, and macrophages and the specialized tissues that containthem. The lymph nodes, lymphatics, and spleen are physical structuresthat housing many of the cells of the immune system. In addition, otherimmune system cells are found in non-lymphoid tissues and in blood. Acharacteristic of the immune system is that it responses to a firstexposure to an antigen (primary response) in a set fashion but thenresponds more strongly and more quickly to a second exposure of anantigen (secondary response), which is a manifestation of immunologicalmemory. The immune system responds to infectious agents and cancer byproducing cells and effector molecules that kill the offendinginfectious agent or cancer cells. Among the cells that kill theattackers are T cells including CD4+ and CD8+ T cells. B cells makeantibodies that can neutralize the infectivity of many infectiousagents. T cells, monocytes, macrophages, and dendritic cells can makeinterferons that interfere with the replication of certain viruses.

“Multimerization scaffold” refers to a molecular structure that confersupon the molecule into which it is incorporated an overall structurethat is operatively linked to two or more TNFSF receptor bindingdomains, such that contact with the multimerized molecule leads toclustering of the complete TNFSF receptor in the membrane of aresponding cell and thereby activates some or all of the functionalpotential of the responding cell. A key concept of the instant inventionis that a many-trimer form a TNFSF ligand is needed to stimulate areceptor-bearing responding cell. For example, structural studies of theGITRL/GITR interaction indicate that two closely localized trimers ofGITRL are needed to bring together or “cluster” two complete GITRreceptor (3 chains of GITR each) (Zhou et al., Proc Natl Acad Sci USA.2008; 105(14):5465-70). A multimerization scaffold is a molecularstructure that provides for this close localization of 2 or more TNFSFreceptor binding, typically 2 or more TNFSF ligand extracellular domains(ECD). In the instant invention, portions of collectins such as SPD orportions of C1q family members such as Acrp30 are used to make singlepolypeptide chains that self-assemble into multimerization scaffolds.Preference is shown for multimerization scaffolds that have “arms”capable of being operatively linked to TNFSF ECD trimers. Alternativeembodiments are contemplated, such as multimerization scaffold that isoperatively linked to single-chain antibodies that bind to a TNFSFreceptor.

“Operatively linked” refers to a method for joining two molecules. Forpolypeptides, this is preferably by a peptide bond, typically achievedby constructing a DNA or RNA template encoding the operatively linkedfusion protein and then expressing the DNA or RNA in a cell or by an invitro method. In some case, chemical crosslinkers can be used toconstruct multimeric forms of TNFSF receptor binding agents as describedin U.S. Pat. No. 6,482,411 B1 which is incorporated by reference.

“TNFSF” refers to a ligand in the Tumor Necrosis Factor (TNF)SuperFamily. See URL http://www.genenames.org/genefamilies/TNFSF for alisting of TNFSFs and their gene names. The TNFSFs are produced astrimeric Type II membrane molecules meaning that their N-terminus pointsinside the cell and their C-terminal end is extracellular, which is thereverse of most cell surface proteins. This makes these proteins verychallenging to engineer using traditional fusion protein strategies.

“TNFSF receptor binder” refers to a molecular fragment that binds to aTNFSF receptor. Exemplary TNFSF receptor binders (or binding domains)include the extracellular domain (ECD) of a TNFSF trimeric molecule orthe receptor-binding portion of an antibody recognizing a TNFSFreceptor. For a receptor-binding portion of an antibody, preference isgive to single-chain antibody constructs (Ahmad et al., Clin DevImmunol. 2012; 2012:980250. PMCID: 3312285). Exemplary TNFSF memberswhose extracellular domains can be used as TNFSF receptor bindersinclude CD40L (TNFSF5), CD27L (TNFSF7), CD137L (TNFSF9), OX40L (TNFSF4),GITRL, 4-1BBL, RANKL, LIGHT, CD70, and BAFF.

“Tumor antigens” refers to proteins, carbohydrates, or lipids found ontumor cells against which the immune system can launch an attack. For adiscussion of tumor antigens, see Kvistborg et al (Curr. OpinionImmunol. 25:284-290, 2013) and Cheever et al. (Clin Cancer Res 15,5323-5337, 2009). Also contemplated as tumor antigens are antigenicpeptides deduced from next-generation sequencing from the RNA or DNA oftumors, including exome sequencing (Segal et al., Cancer Res. 2008;68(3):889-92; Castle et al., Cancer Res. 2012; 72(5):1081-91).

DETAILED DESCRIPTION OF THE INVENTION

This invention describes, inter alia, molecules comprising fusionproteins and the nucleic acids that encode them in which the followingprotein coding domains are operably linked in the following order: ascaffold comprised of a portion of a collectin or C1q family protein orcombinations of dimerizing/trimerizing motifs, an antigen (eitherfollowing the scaffold or contained within the scaffold), and theextracellular domain of a TNF superfamily ligand. An exemplary fusionprotein or nucleic acid that encodes it comprises the antigen,surfactant protein D (SPD) without its carbohydrate receptor domain, andthe extracellular domain of CD40 ligand. Alternatives to surfactantprotein D can also be used, including using immunoglobin (Ig), Acrp30, aGCN4 multimerization motif, or similar proteins as scaffolds for CD40ligand, other members of the TNF superfamily ligands, or other ligandsor receptors, including gp96 or MHC molecules. In one embodiment, themolecules, compositions and/or fusion proteins of the invention do notcontain portions of avidin or streptavidin.

These fusion proteins are designed to allow the targeting of dendriticcells, macrophages, B cells or other antigen presenting cells with theantigen as well as providing necessary activation signals to inducematuration of the targeted dendritic cell, macrophage, B cell or otherantigen presenting cell. This results in the optimal presentation of theantigen to the immune system, and a potent immune response in thetreated individual, either T cell mediated or antibody mediated.

In more detail, the instant invention provides a solution for theproblem of vaccinating against infectious agents and for cancerimmunotherapy. It provides a way to link an adjuvant in the TNFSuperFamily (TNFSF) to an antigen such that the TNFSF adjuvant andantigen arrive at the same cell at the same time. In the case of CD8+ Tcell responses, it is important to provide antigen to dendritic cells(DCs) and other antigen-presenting cells such that the protein antigenis processed by cleavage into peptides and loaded onto MHC-I forcross-presentation on the cell surface as pMHC-I complexes which in turnstimulates the T cell receptor (Signal 1). It is preferable to targetthe antigen to the CD40 receptor on DCs since this results in superiorcross-presentation by a larger number of DC subtypes (Chatterjee et al.,Blood. 2012; 120(10):2011-20). In addition, it is important to activatethe DC that is presenting antigen in order that the DCs present theantigen-specific T cell with accessory signals (Signal 2 and Signal 3).If the DCs display only pMHC-I and are not activated to present othersignals, then the resulting antigen-specific CD8+ T cell becomestolerant and lacks protective effective functions (Bonifaz et al., J ExpMed. 2002; 196(12):1627-38. PMCID: 2196060). Stimulation of the CD40receptor on DCs activates the DCs to provide these other signals andleads to profound CD8+ T cell responses (Bonifaz et al., J Exp Med.2004; 199(6):815-24). Thus, the instant invention provides a strongvaccine for CD8+ T cells by fusing antigen to previously describedmultimeric forms of CD40L comprised of the extracellular domain (ECD) ofCD40L fused to multimerization scaffolds employing portions ofsurfactant protein D (SPD) or Acrp30.

Activation of DCs and other APCs is best performed by a many-trimer formof CD40L where 2 or more trimers are needed to cluster and therebyactivate the CD40 receptors on DCs, as depicted in FIG. 1.

The new understanding of agonistic anti-TNFSF receptor antibodies isshown in FIG. 2. In this case, the antibody is first bound to anadjacent cell via its Fc portion which binds to the Fc receptors on theadjacent cell type (Li and Ravetch, Science. 2011; 333(6045):1030-4.PMCID: 3164589; Wilson et al., Cancer Cell. 2011; 19(1):101-13; White etal., J Immunol. 2011; 187(4):1754-63). This leads to two problems: (1)DCs and other APCs that are not adjacent to an FcR-bearing cell cannotbe stimulated; and (2) if the antibody binds to certain FcRs, then it ispossible that the adjacent cell will kill the DC by antibody-dependentcellular cytotoxicity (ADCC) or phagocytose the DC and eliminate it(Bulliard et al., J Exp Med. 2013; 210(9):1685-93. PMCID: 3754864). Thelater phenomenon may explain the severe depletion of CD40 B cells whenan antibody against CD40 was tested in humans with cancer (Vonderheideet al., J Clin Oncol. 2007; 25(7):876-83). These considerations set thestage for a new and better way to provide both antigen and CD40stimulation to DCs and other APCs.

Another approach was taken by Xiang et al. (J Immunol. 2001;167(8):4560-5) who made a fusion protein of tumor antigen (CEA) joinedto the C-terminal end of CD40L (U.S. Pat. No. 7,279,464 B2; U.S. Pat.No. 6,923,958 B2). However, because the CD40L moiety is not located onthe end of the protein, it could conceivably have impaired binding ofthe ligand to the CD40 receptor. No data were presented to rule out thisconcern, but the vaccine's effectiveness was modest.

In a related approach, Zhang et al. (Proc Natl Acad Sci USA. 2003;100(25):15101-6) fused a tumor antigen onto the N-terminus of the CD40Lextracellular domain and delivered this construct using an adenovirusvector. In this case, the molecular design allowed for CD40L to bindunimpaired to its receptor. Even so, the effectiveness of this vaccinewas relatively modest. This is expected when a 1-trimer form of CD40L isused rather than a receptor-clustering multi-trimer construct such asSPD-Gag-CD40L.

Another approach was taken by Shirwan et al. who produced a fusionprotein between the “core” region of bacterial streptavidin protein(CSA) and the extracellular domain of CD40L or 4-1BBL, as disclosed inU.S. Pat. No. 8,017,582 B2 and in Schabowsky et al., Exp Mol Pathol.86:198-207, 2009. In this case, the N-terminal half of the fusionproteins consisted of CSA where streptavidin naturally assembles into a4-chain molecule. This multimerism pulls together the covalently linkedECDs for CD40L or 4-1BBL. Since streptavidin binds to biotin and sinceproteins can be easily biotinylated, it was possible to biotinylateantigens such as chicken ovalbumin (OVA) or the tumor antigens E7 fromHPV which allows them to bind non-covalently to CSA-CD40L or CSA-4-1BBL.However, in order to be active, CD40L must be used in a multi-trimerform that clusters together two or more CD40 receptors, as depicted inFIG. 1 of the instant application. The relative inactivity of a singletrimer form of CD40L was demonstrated by Haswell et al. (Eur J Immunol.31:3094-3100, 2001; see FIG. 3). In contrast, the CSA-CD40L forms asingle trimer of CD40L, as depicted in FIG. 1B of Schabowsky et al.,which is not desirable from the perspective of efficient receptorstimulation. Furthermore, the biotin-streptavidin interaction in thedesign of Shirwan et al. is non-covalent. The antigen has beenbiotinylated which then allows it to bind to the streptavidin moiety inthe CSA-CD40L complex. However, in vivo, there is free biotin present inbiological fluids that can interfere with the formation of theCSA-CD40L/biotin-antigen complex or induce its dissociation. Incontrast, the instant invention utilizes antigen that has beencovalently joined to CD40L by virtue of the peptide bonds that make upthe SPD-antigen-CD40L fusion protein and thus the protein is notsusceptible to dissociation in the presence of free biotin. Anotherimportant difference is that CSA is a xenogenic protein from bacteriathat is highly antigenic in humans and other vertebrates (Meyer et al.Protein Science 2001; 10(3):491-503; Yumura et al., Protein Science2013; 22(2):213-21). In contrast, the fusion proteins of the instantinvention can be constructed with primarily non-xenogenic proteinssequences such that the only major foreign protein component is theantigen selected for immunization. Therefore, in one embodiment of thepresent invention, the multimerization scaffold and the plurality ofTNFSF receptor binder do not contain any xenogenic portions.

Another system for producing many-trimer forms of OX40L was described byWeinberg et al. in U.S. Pat. No. 7,959,925 B2, which is incorporated byreference. In this system, fusion proteins are made by using anN-terminal immunoglobulin Fc domain which naturally dimerizes viainterchain disulfide bonds. When this is joined to a trimerizing domainwhich is then joined to a TNFSF extracellular domain, it results in whatis described as a hexamer or “dimer of trimers”. In the instantinvention, SEQ ID NO:7 and SEQ ID NO:8 disclose a fusion protein thatprovides for a 2-trimer form of APRIL fused to the HIV-1 Env proteinwhich is expected to elicit a strong antibody response to HIV-1. Theskilled artisan will easily see how the extracellular domain of APRILcould be replaced by the extracellular domain of any other TNFSF ligand,and also how the HIV-1 Env antigen could be replaced by other antigensof interest. Such antigen-multimeric TNFSF fusion proteins are claimedby the instant invention. In addition, the skilled artisan couldenvision other dimerizing domains (such as that from CD4 or CD8) orother trimerizing domains (such as those from GCN4, TRAF2,thrombospondin 1, Matrilin-4, CMP (Matrilin-I), HSFI, or cubulin, asdescribed in U.S. Pat. No. 7,959,925 B2) or the trimerizing domain fromthe SPD “neck” region in U.S. Pat. No. 6,190,886, which is incorporatedby reference.

As described in the instant application, a surprisingly active vaccinecan be made by incorporating an antigen with the arms of SPD in the4-trimer SPD-CD40L construct that was previously developed by theinventors and shown in FIGS. 3 and 4. For demonstration purposes, theHIV-1 Gag antigen was inserted into the coding region for the SPDcollagen-like arm as shown in SEQ ID NO:1 and SEQ ID NO:2 and depictedin FIG. 5. This fusion protein uses the natural SPD “arm” which has beenshown to be 46 nm long in shadow electronmicroscopic studies. Thecollagen-like triple helical structure and results from the classGly-Xaa-Yaa collagen-like repeats in the protein which number 59 repeatsin the arm. For the instant invention, the length of this arm can bevaried in two ways: (1) Amino acid deletions can be introduced thattruncate one Gly-Xaa-Yaa motif; and (2) the antigen can be insertedvariably along the length of the arm. Considering the 177 amino acids inthe 59 collagen-like repeats, the antigen domain can be positioned from10 to 177 amino acids more C-terminal from the hub, or preferably from20 to 140 amino acids more C-terminal from the hub, or more preferablyfrom 40 to 120 amino acids more C-terminal from the hub. Likewise, theantigen domain can placed closer or further from the TNFSF extracellulardomain (ECD). For example, the antigen domain could be from 0 to 167amino acids more N-terminal from the TNFSF ECD, or more preferably from40 to 120 amino acids more N-terminal from the ECD. As non-limitingexamples, SEQ ID 13 and SEQ ID 14 show a fusion protein where theantigen domain was shifted by 10 amino acid positions within the arm ofSPD. Likewise, SEQ ID NO 17 and SEQ ID NO 18 show a fusion protein inwhich 20 amino acids have been removed from the SPD arm. With this as aguide, the skilled artisan will know that it is not critical exactlywhere in the SPD arm the antigen domain should be positioned.

As previously described by the inventors, 2-trimer forms of TNFSFligands can be made using Acrp30. FIGS. 3 and 4 show the design andvaccine adjuvant efficacy of an Acrp30-CD40L fusion protein. Thismolecule has two collagen-like arms. Accordingly, it is contemplated toplace an antigen domain within the arms of Acrp30 as shown in SEQ ID 3and SEQ ID 4 which place the HIV-1 Env antigen within the arms of anAcrp30-BAFF fusion protein. Analogous fusion proteins could be made fromother collectin fusion proteins besides SPD-TNFSFs and from other C1qfamily molecules besides Acrp3-TNFSFs.

A feature of these fusion proteins is that they can readily be madeusing the natural collectin or C1q family sequences and TNFSF sequencesfrom a variety of organisms. It is preferable to use the murine codingsequences for studies in mice, the macaque coding sequences for studiesin macaques, the human coding sequences for use in humans, etc. Asnon-limiting examples, the sequences shown provide fusion protein madeusing either murine or human sequences. Thus, animal vaccine uses arespecifically contemplated as one use of the instant invention.

In these cases, antigen was introduced into many-trimer forms of TNFSFsby standard genetic engineering methods familiar to the skilled artisan.Such fusion proteins can be made by ligating together segments of genesor, more preferably, by ordering a custom synthesis from a commercialsupplier (e.g. DNA2.0, Genset, Genewiz, and other suppliers). In othercases, it is possible to prepare antigenic peptides and TNFSF trimersseparately and then link them together by chemical methods. The linkingreagents and synthesis strategies that can be used are described in U.S.Pat. No. 6,482,411 B1, which is incorporated by reference.

There is a wide choice of antigens from infectious disease antigens,depending on the species in need of vaccination. Without limitation,these can be selected from the following list of disease-causingpathogens:

Viruses such as influenza A and B, parainfluenza, poxviruses, ebolavirus, hepadnavirus, filoform viruses such as marburg virus, denguefever virus, influenza A and B, respiratory syncytial virus, measles(rubeola virus), human immunodeficiency virus (HIV), humanpapillomavirus (HPV), varicella-zoster, herpes simplex I and 2,cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus,rhinovirus, adenovirus, orthomyxovirus, papillomavirus, parvovirus,picornavirus, poliovirus, mumps, rabies, reovirus, rubella, togavirus,retrovirus, coxsackieviruses, equine encephalitis, Japaneseencephalitis, yellow fever, Rift Valley fever virus, hepatitis A, B, C,D, and E virus, hantavirus, coronavirus (including SARS and MERS), andthe like;

Microbial agents such as Borrelia species, Bacillus anthracis, Borreliaburgdorferi, Bordetella pertussis, Camphylobacter jejuni, Chlamydiaspecies, Chlamydial psittaci, Chlamydial trachomatis, Clostridiumspecies, Clostridium tetani, Clostridium botulinum, Clostridiumperfringens, Corynebacterium diphtheriae, Coxiella species, anEnterococcus species, Erlichia species Escherichia coli, Francisellatularensis, Haemophilus species, Haemophilus injiuenzae, Haemophilusparainjiuenzae, Lactobacillus species, a Legionella species, Legionellapneumophila, Leptospirosis interrogans, Listeria species, Listeriamonocytogenes, Mycobacterium species, Mycobacterium tuberculosis,Mycobacterium leprae, Mycoplasma species, Mycoplasma pneumoniae,Neisseria species, Neisseria meningitidis, Neisseria gonorrhoeae,Pneumococcus species, Pseudomonas species, Pseudomonas aeruginosa,Salmonella species, Salmonella typhi, Salmonella enterica, Rickettsiaspecies, Rickettsia ricketsii, Rickettsia typhi, Shigella species,Staphylococcus species, Staphylococcus aureus, Streptococcus species,Streptococccus pnuemoniae, Streptococcus pyrogenes, Streptococcusmutans, Treponema species, Treponema pallidum, a Vibrio species, Vibriocholerae, Yersinia pestis, and the like;

Fungal, protozoan, and parasitic agents such as Aspergillus species,Candida species, Candida albicans, Candida tropicalis, Cryptococcusspecies, Cryptococcus neoformans, Entamoeba histolytica, Histoplasmacapsulatum, Coccidioides immitis, Leishmania species, Nocardiaasteroides, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii,Trichomonas vaginalis, Toxoplasma species, Trypanosoma brucei,Schistosoma mansoni, Pneumocystis jiroveci, and the like.

There is a wide choice of tumor antigens, depending on the species inneed of cancer immunotherapy. Without limitation, these can be selectedfrom the following list of cancer-associated antigens:

gp100; WT1; Melan-A; tyrosinase; PSMA; HER-2/neu; MLC-l; PRAME;topoisomerase; BRAF V600E; bcr-Abl; sialyl-Tn; carcinoembryonic antigen;ErbB-3-binding protein-I; alpha-fetoprotein; and the cancer testisantigens MAGE-AI, MAGEA4, and NY-ESO-1; MART-1, Dipeptidyl peptidase IV(DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b,Colorectal associated antigen (CRC)-CO17-1 AlGA 733, CarcinoembryonicAntigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amll,Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1,PSA-2, and PSA-3, prostate specific membrane antigen (PSMA), MAGE-familyof tumor antigens (e.g., MAGEAI, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-AS, MAGE-A9, MAGE, MAGE-Xp2 (MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4,MAGEC5), GAGE-family of tumor antigens (e.g., GAGE-I, GAGEIn 2, GAGE-3,GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGES, GAGE-9), BAGE, RAGE, LAGE-1, NAG,GnT, MUM-1, CDK4, tyrosinase, p53, MLC family, HER2/neu, p21ras, RCAS1,a-fetoprotein, E-cadherin, alpha-catenin, beta-catenin andgamma-catenin, p 120ctn, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL), EGFRviii, SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27,adenomatous polyposis coli protein (APC), fodrin, PI A, Counexin 37,Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such ashuman papilloma virus proteins, Smad family of tumor antigens, LMP-1,LMP-2, EBV-encoded nuclear antigen (EBNA)-1, or c-erbB-2, and the like.

There is a wide choice of delivery methods for the vaccines of theinstant invention. Where the vaccine is comprised of a nucleic acidsequence, it can be delivered using a DNA or RNA vectors. Withoutlimitation, these can be selected from the following list: Adenovirus(as shown in FIG. 12 for example), poxvirus including Modified VacciniaAnkara, Herpesviruses, retroviruses, lentiviruses, Newcastle DiseaseVirus, Mumps Virus, Measles Virus, Vesicular Stomatitis Virus,rhabdovirus, Para-influenza Virus, Sendai virus, Influenza Virus,Reovirus, and a Seneca Valley virus, alphavirus, Sindbis virus,Venezuealan Equine Envephalitis (VEE), Coxsackie virus, myxoma virus,viral organisms include those that are dsDNA viruses, ssDNA viruses,dsRNA viruses, (+) ssRNA viruses (−) sRNA viruses, ssRNA-RT viruses, anddsDNA-RT viruses, and the like.

Vaccines of the present invention can also be delivered as plasmid DNAsthat include a promoter (e.g., CMV promoter) and a transcriptiontermination and polyadenylation sequence. Such plasmids also includegenes needed for growth in bacteria, but fragments of DNA can also beprepared by in vitro enzymatic synthesis. An exemplary plasmid used inthe experiments in FIGS. 4 and 6-17 is pcDNA3.1 (Life Technologies,Inc., Carlsbad, Calif.) but other choices are available. The DNA can bedelivered directly by injection into muscle (“naked” DNA vaccination) asshown in FIGS. 8-11 and 15-17. It can also be delivered by a number ofmeans including electroporation, microinjection, gene gun delivery,lipofection, polymer-mediated delivery, and the like. The same methodscan be used for RNA vaccination. In addition, for bacteria that entercells such as Salmonella or Listeria, plasmid DNA can be introduced intothese bacteria which then carry that DNA into the eukaryotic host cell,a process called “bactofection.”

As another use of the instant invention, fusion proteins comprised of anantigen linked to a many-trimer TNFSF can be administered to APCs likedendritic cells ex vivo, as shown in FIGS. 7 and 14. Once the antigenhas been delivered and the APCs activated, these DCs can then bedelivered to a host as a cellular form of vaccination (Barth et al.,2010; 16(22):5548-56. PMCID: 2994719).

Without limitation, the following examples of invention are disclosed:

EXAMPLE 1 Vaccination to Elicit CD8+ T Cells Against HIV-1 Gag Antigen

CD40 ligand (CD40L, CD154) is a membrane protein that is important forthe activation of dendritic cells (DCs) and DC-induced CD8+ T cellresponses. To be active, CD40L must cluster CD40 receptors on respondingcells. To produce a soluble form of CD40L that clusters CD40 receptorsnecessitates the use of a multi-trimer construct. With this in mind, atripartite fusion protein was made from surfactant protein D (SPD),HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffoldfor the multi-trimer protein complex. This SPD-Gag-CD40L proteinactivated CD40-bearing cells and bone marrow-derived DCs in vitro.Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination ofmice with pSPD-Gag-CD40L induced an increased number of Gag-specificCD8+ T cells with increased avidity for MHC-I-restricted Gag peptide andimproved vaccine-induced protection from challenge by vaccinia-Gagvirus. The importance of the multi-trimeric nature of the complex wasshown using a plasmid lacking the N-terminus of SPD that produced asingle trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was onlyweakly active on CD40-bearing cells and did not elicit strong CD8+ Tcell responses or improve protection from vaccinia-Gag challenge. Anadenovirus-5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much strongerthan Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection(i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, theseresults show the potential of a new vaccine design in which antigen isintroduced into a construct that expresses a multi-trimer soluble formof CD40L, leading to strongly protective CD8+ T cell responses.

DNA vaccination induces both cellular and humoral responses against anencoded antigen, protecting animals against subsequent infection with amicrobial pathogen. DNA vaccines are potent inducers of virus-specific Tcell responses and studies have shown that prophylactic DNA vaccines,administered either alone or with recombinant viral vaccines asprime/boost vaccine, can provide protection against challenge with viralpathogens including SIV. The HIV-1 Gag antigen encoded within DNA orviral vector vaccines is known to induce measurable immune responses,providing a method to vaccinate against HIV-1. One strategy to enhancethe effectiveness of DNA vaccines encoding weakly immunogenic antigensis by co-delivering genes encoding molecular adjuvants. TNF superfamilyligands (TNFSFL) including CD40L are costimulatory molecules involved indendritic cell (DC) and T cell activation and have previously beentested as adjuvants to enhance immune responses in several vaccinationstudies.

CD40L acts on DCs to induce or “license” CD8+ T cell responses. CD40Lalso works on DCs to diminish the immune suppression due toCD4+CD25+FoxP3+ regulatory T cells (Tregs) and prevents the prematuredisappearance vaccine-generated CD8+ T cells. Consequently, we andothers have examined the potential of CD40 stimulation as an adjuvantfor vaccines designed to generate CD8+ T cell responses.

CD40-mediated activation requires clustering of this receptor leading tothe assembly of a supramolecular signaling complex inside cells. WhenCD40L is expressed on CD4+ T cells, the array of membrane CD40Lmolecules ligates receptors on DCs and other cells to create a patch ofclustered CD40 receptors that activates downstream events. For solubleligands of CD40, some other way must be found to induce CD40 receptorclustering. Most reports on CD40 activation use agonistic anti-CD40antibodies. It is now recognized that these antibodies only induce aCD40 signal if they are mounted onto Fc receptors (FcRs), therebycreating an array of anti-CD40 antibodies that can cluster the receptorson an adjacent CD40 receptor-bearing cell. This requirement restrictsthe effectiveness of anti-CD40 antibodies to tissue microenvironmentsthat contain FcR-bearing cells. Other drawbacks of using anti-CD40antibodies are their propensity to generate host antibodies againstthemselves, their toxicity for mice and humans, and their depletingeffect on CD40-bearing B cells in the blood. These negative qualitiesargue against the routine use of agonistic anti-CD40 antibody as anadjuvant for vaccines given to otherwise healthy people in order toprevent infection by pathogens such as HIV-1.

The use of CD40L presents an alternative to agonistic anti-CD40antibodies as a vaccine adjuvant. CD40L is made as a Type II membraneprotein but can be proteolytically cleaved from the cell surface andreleased as a soluble single trimer. By itself, a single trimer of CD40Lis unable to provide clustering of CD40 receptors sufficient to generatea cell signal. Consequently, we devised fusion proteins in which theextracellular domain of CD40L is joined to a scaffold protein such assurfactant protein D (SPD). The resulting fusion protein, SPD-CD40L, isexpected to form a plus sign-shaped 4-trimer molecule held together atits N-terminal “hub” by interchain cysteine bonds. Each “arm” of the SPDportion is a collagen-like triple helix that presents the CD40L trimerson the outside of the molecule for easy interaction with CD40 receptors.As expected, we previously found that SPD-CD40L activated DCs in vitroand was a strong vaccine adjuvant for CD8+ T cell responses againstHIV-1 antigens.

In the previous study, mice were vaccinated with plasmid DNAs for HIVantigens such as Gag (pGag) mixed in a single syringe with pSPD-CD40L.In the present study, we considered the effects of introducing the HIV-1Gag antigen into the SPD-CD40L protein to create SPD-Gag-CD40L, a singlechain peptide that retains the ability to form a multi-trimer structurecapable of clustering and thereby activating the CD40 receptor. Thismolecular design resulted in a DNA vaccine that elicited much strongerGag-specific CD8+ T cell responses capable of protecting mice fromchallenge by vaccinia virus engineered to express Gag (vaccinia-Gag).Since DNA vaccination is relatively inefficient, viral delivery was alsoexamined by introducing SPD-Gag-CD40L into an adenovirus-5 (Ad5) vaccinevector. The resulting Ad5-SPD-Gag-CD40L vaccine provided essentiallytotal protection from vaccinia-Gag challenge, further attesting to theremarkable effectiveness of including the antigen inside the SPD-CD40Lconstruct rather than administering SPD-CD40L as a separate adjuvantmolecule.

Materials and Methods Construction and Preparation of DNA Plasmids

To construct a HIV-1 Gag DNA vaccine (pGag), the gag coding sequence wasfused with the first 21 amino acids of human tissue plasminogenactivator (t-PA) as a signal peptide as described previously (Stone etal., J Virol. 2006; 80(4):1762-72). A DNA construct encoding murineSPD-CD40L was also previously described (Stone et al., J Virol. 2006;80(4):1762-72). To construct SPD-Gag-CD40L, the p55 gag sequence frompGag was inserted into the “arm” portion of murine SPD between aminoacids 105 and 106 within the construct SPD-CD40L (i.e. between peptidesequence GERGLSG and PPGLPGI of murine SPD) (see FIG. 5). To constructpTrimer-Gag-CD40L, the ScGag coding sequence was fused with amino acid106 of mouse SPD within construct SPD-CD40L (i.e. fusing ScGag to afragment of SPD-CD40L starting at peptide sequence PPGLPGI), therebydeleting the N-terminal portion of SPD that contains thedicystine-containing “hub” region needed for self-assembly into a4-armed molecule. As a result, this construct is expected to form singletrimers of Gag-SPD-CD40L (see FIG. 5). Plasmid pIL-12p70, encoding mousesingle chain IL-12, was purchased from Invivogen Inc. All plasmids werepropagated in Escherichia coli strain TOP10. Endotoxin-free DNA plasmidpreparations were prepared using an Endofree Giga plasmid kit (Qiagen).Plasmids were further purified to remove residual endotoxins withadditional Triton-X114 extractions as previously described (Stone etal., J Virol. 2006; 80(4):1762-72). Plasmid endotoxin level was <0.2EU/ml for all constructs as confirmed by LAL endotoxin assay (LonzaInc.). Gag protein secretion for all Gag-containing constructs wasconfirmed by p24 ELISA assay on supernatants from transfected 293Tcells.

Transient Transfection and Western Blotting of Protein Constructs

293T cells were transiently transfected with plasmid constructs usingGenjet-plus Transfection Reagent (Signagen Laboratories, Iamsville,Md.). A control transfection with GFP plasmid was used to confirmtransfection efficiency of each reaction. Forty-eight hours later,supernatants were centrifuged and filtered with a 0.45 μm filter toremove debris. Filtered supernatant was reduced with 2-mercaptoethanol,loaded onto sodium-dodecyl sulfate-10% polyacrylamide gels (10%SDS-PAGE) (BioRad), electrophoresed, and blotted onto PVDF membranes(Pierce). The membranes were blocked using 5% (w/v) dry milk and thenprobed with goat anti-mouse CD40L antibody (R&D Systems), followed byincubation with anti-goat horseradish peroxidase-conjugated antibodies(Jackson Immunoresearch). The protein bands were developed onto X-rayfilm using chemiluminescence. To further evaluate high molecular weightcomplexes, a non-denaturing PAGE was performed in the absence of SDS andreducing agent.

CD40 in Vitro Activity Assay

A CD40 receptor-bearing reporter cell line (CD40-293-SEAP) was used tomonitor CD40L-mediated activation. This 293-derived cell lineconstitutively expresses human CD40 receptor along with the gene forsecreted alkaline phosphatase (SEAP) gene under control of NF-κB(Maurais et al., Virology. 2009; 385(1):227-32). Briefly, 80,000CD40-293-SEAP reporter cells, grown in DMEM medium with 10% FBS, wereplated in each well of a 96-well plate. A total of 100 μl ofSPD-Gag-CD40L, SPD-CD40L or pcDNA3.1 transfected 293T supernatant wasadded to the reporter cells for 24 h in triplicate at various dilutions.On the following day, 10 μl/well of the supernatants was added to thewells of a 96-well assay plate together with 100 μl/well of QUANTI-BlueAlkaline Phosphatase substrate (InvivoGen). The plates were incubatedfor 20 min at 20° C. and OD was read at 650 nm.

DC Activation and Maturation Assay

Bone marrow-derived murine DCs were generated by standard methods (Inabaet al., Cellular immunology. 1995; 163(1):148-56) with the followingmodifications: Bone marrow cells were obtained from C57BL/6 mice andwashed in RPMI 1640 media. The cells were then placed in tissue culturetreated T75 flasks at a concentration of 1×106 cells per ml in 20 mlcomplete RPMI (RPMI 1640 with 10% FBS, 20 μg/mlgentamycin sulfate, 50 μM2-mercaptoethanol), and 20 ng/ml murine recombinant GM-CSF and 10 ng/mlmurine recombinant IL-4 (Peprotech, Rocky Hill, N.J.)). Cells werecultured at 37° C., 5% CO2 and on day 3, media was replaced with freshcomplete RPMI containing cytokines. On day 5, cells were harvested andwashed and resuspended in complete RPMI at 5×10E5 cells/ml. A total of 2ml was added to each well of 6-well tissue culture treated plates.Subsequently, 300 μl of supernatant containing SPD-Gag-CD40L or DCactivation cytokine mix (containing TNF, IL-1beta, IL-6, and PGE2) wasadded and cells were incubated for 36 hours. Cells were harvested andstained with hamster anti-mouse CD11c clone N418 PE-Cyanine7 conjugate(eBioscience, San Diego, Calif.) combined with one of the followingantibodies: anti-mouse CD80 clone 16-10A1, anti-mouse CD86 clone GL1,anti-mouse CD40 clone 1C10, anti-mouse CD83 clone Michel-17, anti-mouseMHC Class II (I-A/I-E) clone M5-114.15.2, and anti-mouse CCR7 clone 4B12(all from eBioscience). After flow cytometry analysis, the meanfluorescence intensity for each antibody was calculated for CD11c+dendritic cells under each experimental condition. Flowjo 7.6.4 flowcytometry analysis software (FlowJo, Ashland, Oreg.) was used foranalysis. Three independent wells were analyzed for each condition.

Production of Recombinant Adenovirus Containing Gag Antigen orSPD-Gag-CD40L

The construction of replication-deficient adenovirus (pAdEasy-1)containing codon-optimized Gag with a t-PA signal peptide orSPD-Gag-CD40L was performed as described by the manufacturer (AdEasyAdenoviral vector system, Agilent Technology, Inc.). Briefly, geneconstructs were PCR amplified and cloned into the pAdenoVator-CMV5shuttle vector (Qbiogene). CMV5-shuttle vector clones were confirmed bysequencing and then electroporated into BJ5183 cells containing thepAdEasy-1 plasmid to induce homologous recombination. The recombinedpAdEasy-1 vector was linearized and transfected into AD293 cells(Stratagene). Following propagation in AD293 cells, recombinant Ad5viruses were purified and concentrated using the Adeno-X Megapurification kit (Clontech). The concentration of Ad5 viral particles(vp) was determined by measuring the absorbance at 260 nm and 280 nm,and calculated using the formula vp/ml=OD260×viral dilution×1.1×1012. Todetermine infectious units, viruses were titered using the Adeno-x RapidTiter kit (Clontech).

Mice and Immunization Schedule

Female BALB/c mice (7-8 weeks old) were used in all vaccinationexperiments. Animals were housed at the University of Miami under theguidelines of the National Institutes of Health (NIH, Bethesda, Md.).All animal experiments were performed in accordance with national andinstitutional guidance for animal care and were approved by the IACUC ofthe University of Miami. Different groups of mice were immunized withplasmid DNA or Ad5 viruses for immunological and vaccinia challengeexperiments.

DNA Immunization Schedule: DNA was injected intramuscularly into thequadriceps muscle of both hind limbs. Vaccinations were given threetimes at two-week intervals with 100 μg of SPD-Gag-CD40L or 100 μgGagplasmid mixed with either 20 μg of pcDNA3.1, pSPD-CD40L, or pIL-12p70plasmids. Doses were administered in a total volume of 100 μl PBS (50 μlper limb). Control mice were injected with 100 μg of pcDNA3.1 emptyvector.

Splenocyte preparation: Two weeks following the final DNA immunization,mice were euthanized and spleens were removed. Single cell splenocytepreparations were obtained by passage through a 40 μm nylon cellstrainer (BD Falcon). Erythrocytes were depleted with lysis buffer(Sigma) and splenocytes washed thoroughly using R10 media (RPMI 1640supplemented with 10% fetal bovine serum (FBS), 50 μM2-mercaptomethanol, 100 U/ml of penicillin, 100 μg/ml streptomycin, and10 mM HEPES).

Adenovirus Immunization Schedule: Five mice per group were immunized byintramuscular injection with Ad5 constructs twice at a two-weekinterval. Viral vector was injected in a total volume of 100 μl PBS (50μl per limb) in the quadriceps muscles of both hind limbs.

Enzyme Linked Immunospot (ELISPOT) Assay

IFN-γ and IL-2 ELISPOT assays were performed to determine antigenspecific cytokine secretion from immunized mouse splenocytes. ELISPOTassays were carried out per manufacturer's protocol (R&D Systems) using96-well MAIP plates (Millipore). Freshly prepared vaccinated mousesplenocytes (1×105 cells/well) were added to each well of the plate andstimulated for 18 h at 37° C., 5% CO2 in the presence of HIV-1 Gagpeptide AMQMLKETI (10 μg/ml or as described). A c-myc peptide (negativecontrol) and PMA/Ionomycin (positive control) were evaluated tocalculate the minimum and maximum number of antigen-specific ELISPOTsrespectively. After 18 h, spots were developed with AEC substrate kit(Vector Laboratories) according to manufacturer's instructions. Themembrane was read by automated plate reader (CTL Immunospot) forquantitative analyses of the number of IFN-γ or IL-2 spots formingcounts (SFC) per million cells plated, subtracting negative controlvalues.

T Cell Receptor Avidity ELISPOT Assay

ELISPOT was performed as described, stimulating the cells with 1 μg/ml,10-3 μg/ml, or 10-5 μg/ml of Gag peptide (AMQMLKETI) to evaluate thenumber of T cells able to secrete IFN-γ at limiting peptideconcentrations.

ELISA Assay for Anti-Gag IgG Responses

Anti-Gag antibody production was measured by ELISA assay. HIV-1 p55 Gagprotein (10 μg/ml) was coated onto 96-well ELISA plates overnight at 4°C. Mouse sera at varying dilutions (1:30, 1:120, 1:480 and 1:1,920) wereadded to Gag-coated wells and incubated at room temperature for 2 h withshaking. After the plates were washed, Gag antigen specific IgGantibodies were detected using alkaline phosphatase-conjugated goatanti-mouse IgG (Jackson Immunoresearch Inc.). Signal was developed usingBluePhos substrate (KPL, Inc.). Plates were analyzed using a 96-wellplate absorbance reader at 650 nm. Endpoint titers were calculated asthe highest dilution with more than twice the background absorbance ofcontrol wells.

Vaccinia-Gag Virus Challenge

Two weeks following DNA or Ad5 immunization, mice were challenged i.pwith 1×107 vp vaccinia-gag virus vP1287 as described (Qiu et al., JVirol. 1999; 73(11):9145-52). Five days following challenge, mice weresacrificed and ovaries were removed and homogenized in 500 μl PBS. Formeasurement of virus titers, samples were sonicated and evaluated intriplicate by 10-fold serial dilution on Vero cells plated in 24 wellplates. Following 48-hour incubation, the plates were stained with 0.1%(w/v) crystal violet in 20% ethanol. Plaques were counted and expressedas the plaque-forming units (PFU) of virus in total lysate volume(PFU/mouse).

Statistical Analysis

All error bars represent standard error from the mean. Graph pad Prism6.0 software was used to calculate significance by one way ANOVA formultiple comparison or by two-tailed Student's t test, comparing micevaccinated with SPD-Gag-CD40L, Gag, or Gag antigen+adjuvant (SPD-CD40Lor IL-12p70). In all figures, p values are labeled by asterisks denotingp<0.05 (*), p<0.01 (**), and p<0.001 (***). Any unlabeled comparisonswere not statistically significant between groups.

Results Construction and Expression of Multi-Trimer SPD-Gag-CD40L

CD40L is naturally produced as a Type II membrane protein on the surfaceof activated CD4+ T cells and other cells. When an activated CD4+ T cellcomes in contact with a DC, an immunological synapse forms that clustersCD40 receptors in the DC membrane, which in turn initiates downstreamevents in the DC. To mimic this situation using a soluble CD40L protein,a many trimer form of CD40L is needed since single trimers of CD40L donot provide an effective stimulus (reviewed in Kornbluth et al.,International Reviews of Immunology. 2012; 31(4):279-88). Consequently,multi-trimer soluble forms of CD40L were developed by fusing SPD withthe CD40L extracellular domain, where SPD provides a self-assemblingscaffold for multimerization. SPD-CD40L mimics the multivalent nature ofmembrane CD40L and was previously shown to activate B-cells, macrophagesand dendritic cells in vitro and enhance vaccine responses in vivo. Inthe previous vaccine studies, antigen and multi-trimer CD40L adjuvantwere used as separate molecules and mixed together for immunization(Stone et al., J Virol. 2006; 80(4):1762-72). To further improve thisvaccine design, an immunogen was developed that incorporated antigen(exemplified by HIV-1 Gag) and multi-trimer CD40L into a singlepolypeptide, SPD-Gag-CD40L. The p55 portion of Gag was inserted intoprotein sequence for the collagen-like trimeric “arm” of SPD, betweenamino acid 105 and 106 of mouse SPD within the SPD-CD40L construct (FIG.5A). To show that SPD-Gag-CD40L has the expected structure, protein wasproduced by transfecting 293T cells with pSPD-Gag-CD40L plasmid DNA.Using reducing conditions, SDS-PAGE, and western blotting for CD40L, theresulting culture supernatant was found to contain a single protein ofthe expected size of 105 kDa (FIG. 6A). A single 105 kDa band was alsoobserved using antibody to the p24 portion of Gag (data not shown). Toconfirm that SPD-Gag-CD40L forms a large protein complex, PAGE andwestern blotting were performed using a non-denaturing gel in theabsence of reducing agents. Multiple bands were observed at >200 kDamolecular weight, demonstrating the formation of large multimericcomplexes (FIG. 6B).

Biological Activity of Multi-Trimer Soluble SPD-Gag-CD40L

To assess the ability of SPD-Gag-CD40L to stimulate the CD40 receptor, aCD40-bearing indicator cell line was used as described previously(Maurais et al., Virology. 2009; 385(1):227-32). In this cell line, CD40stimulation activates the NF-κB pathway which in turn activates the κBpromoter driving the expression of secreted alkaline phosphatase (SEAP)that is measured by a colorimetric enzymatic assay. Supernatants from293T cells transfected with pSPD-Gag-CD40L or parent pSPD-CD40Lstimulated these CD40 receptor-bearing cells to produce SEAP (FIG. 7A).In contrast, supernatants from 293T cells transfected with pcDNA3.1empty vector were inactive. To evaluate the biological activity of thesoluble forms of CD40L, bone marrow-derived dendritic cells were treatedwith supernatants from 293T cells transfected with either pSPD-Gag-CD40Lor pcDNA3.1 empty vector. A cytokine mix (TNF, IL-1beta, IL-6, and PGE2)was used to “mimic” an inflammatory environment and used as a positivecontrol. As shown in FIG. 7B, CD80, CD86 and CCR7 were significantlyupregulated by SPD-Gag-CD40L supernatant compared to pcDNA3.1 controlsupernatant. In contrast, CD40 expression was significantly reduced,consistent with endocytosis of CD40 following SPD-Gag-CD40L ligation.

As a DNA Vaccine, Multi-Trimer Soluble SPD-Gag-CD40L was MoreImmunostimulatory than Separate Plasmids for Gag Antigen and SPD-CD40LAdjuvant

Plasmid DNA for SPD-Gag-CD40L (pSPD-Gag-CD40L) was evaluated for itsability to enhance immune responses as a DNA vaccine. Mice werevaccinated three times at two-week intervals with an intramuscularinjection of 100 μg of pSPD-Gag-CD40L plasmid DNA. For comparison, 100μg of plasmid DNA encoding soluble secreted Gag antigen (pGag) was mixedwith 20 μg of separate plasmids encoding either SPD-CD40L or IL-12p70adjuvants or pcDNA3.1 empty control vector. The vaccination schedule isoutlined in FIG. 8A. Two weeks following the third vaccination, T cellresponses were analyzed by IFN-γ and IL-2 ELISPOT assays using theKd-restricted HIV-1 Gag peptide AMQMLKETI to stimulate mousesplenocytes. As shown in FIG. 8B, there was a significant increase inGag-specific CD8+ T cell responses measured by IFN-γ ELISPOT insplenocytes from mice vaccinated with pSPD-Gag-CD40L compared to micevaccinated with pGag alone or a mixture of separate plasmids for pGagantigen combined with either pSPD-CD40L or pIL-12p70 adjuvants.Comparing pSPD-Gag-CD40L to unadjuvanted pGag alone, mean IFN-gammaELISPOT responses increased >60-fold. In contrast, the responses toseparate plasmids for pGag mixed with pSPD-CD40L or pIL-12p70 adjuvantswere much less. Similarly, IL-2 ELISPOT responses were significantlyincreased for pSPD-Gag-CD40L compared to pGag alone or separate plasmidsfor pGag antigen mixed with pSPD-CD40L or pIL-12p70 adjuvants (FIG. 8C).Comparing pSPD-Gag-CD40L to pGag alone, mean IL-2 ELISPOT responsesincreased >10-fold.

To determine if high avidity CD8+ T cells were present, CD8+ T cellIFN-γ ELISPOT responses were tested at limiting AMQMLKETI peptideconcentrations. As shown in FIG. 9A, pSPD-Gag-CD40L significantlyincreased IFN-γ ELISPOT responses compared to other vaccine groups atall peptide dilutions. At 10 pg/ml of AMQMLKETI peptide, IFN-gammaELISPOT responses were only detectable from the splenocytes of micevaccinated with pSPD-Gag-CD40L. Overall, these data show thatpSPD-Gag-CD40L markedly enhanced anti-Gag CD8+ T cell immune responsesand CD8+ T cell avidity levels compared to alternative vaccinationapproaches.

To evaluate humoral immune responses, Gag-specific IgG antibody titersin mice serum were measured by ELISA assay two weeks followingvaccination. As shown in FIG. 9B, all vaccine groups induced similarGag-specific IgG responses compared to Gag vaccination alone and therewere no significant differences between groups.

Single-Trimer Gag-CD40L Fusion Protein Failed to Enhance ImmuneResponses Compared to Multi-Trimer SPD-Gag-CD40L

We next evaluated the role of multi-trimerization by the SPD scaffold onthe immune response. The N-terminus of SPD is involved in disulfidebonding and is required to form 4-trimer complexes (Crouch et al., JBiol Chem. 1994; 269(25):17311-9). Deleting this N-terminal portion ofSPD (amino acids 106-256 in murine SPD) results in a single-trimer formof Gag-CD40L (pTrimer-Gag-CD40L). A t-PA signal peptide was added at theN-terminus sequence to direct protein secretion, followed by HIV-1 Gag,amino acids 106-256 of murine SPD, and then amino acids 47-260 of murineCD40L. Lacking the multimerizing “hub” of SPD, this construct isexpected to form single trimer molecules containing Gag and CD40L. Toexamine the biological activity of pTrimer-Gag-CD40L, protein was madeby transfecting 293T cells with pTrimer-Gag-CD40L plasmid and testingthe resulting supernatant in the CD40 NF-κB SEAP indicator cell lineassay described above. As expected, with only one trimer of CD40L, thepTrimer-Gag-CD40L-encoded protein had little or no activity in thisassay (data not shown), confirming previous reports that single trimersof CD40L are essentially unable to stimulate CD40 receptor-bearing cells(Holler et al., Mol Cell Biol. 2003; 23(4):1428-40; Haswell et al., MolCell Biol. 2003; 23(4):1428-40). Mice were then vaccinated with DNAvaccines encoding pGag (unadjuvanted antigen alone), pTrimer-Gag-CD40L(single trimer of Gag antigen fused to CD40L) or pSPD-Gag-CD40L(multi-trimer of Gag antigen fused to CD40L). Mice vaccinated withpSPD-Gag-CD40L showed a significant increase in IFN-gamma ELISPOTresponses compared to unadjuvanted pGag alone or pTrimer-Gag-CD40L whichcontains Gag and CD40L but lacks the multi-trimer structure (FIG. 10A).Also observed was a significant increase in IL-2 ELISPOT responses forthe pSPD-Gag-CD40L group vs. pTrimer-Gag-CD40L (FIG. 10B).

Vaccination with pSPD-Gag-CD40L Protected Mice from Virus Challenge byVaccinia-Gag

To determine the protective efficacy of the CD8+ T cells induced by DNAvaccination with pSPD-Gag-CD40L, vaccinated mice were challenged byvaccinia virus expressing the HIV-1 Gag antigen (vP1287 or vaccinia-Gag)(Qiu et al., J Virol. 1999; 73(11):9145-52). Two weeks following finalDNA vaccination, mice were challenged intraperitoneally withvaccinia-gag (10E7 PFU). As shown in FIG. 11A, mice vaccinated withpSPD-Gag-CD40L had a significantly less tissue virus in ovaries comparedwith unvaccinated animals (p<0.001) or animals vaccinated with pGag DNAvaccine alone (p<0.05) when vaccinia PFUs were measured on day 5following vaccinia-Gag challenge. Overall, 4 out of 13 mice vaccinatedwith pSPD-Gag-CD40L had undetectable viral titers (less than 10 PFU intotal ovary lysate).

To determine the effect of CD40L multi-trimerization on the protectionconferred by vaccination, mice were vaccinated with pcDNA3.1 emptyvector, pGag antigen alone, pTrimer-Gag-CD40L, or pSPD-Gag-CD40L (FIG.11B). There were no significant differences in vaccinia-Gag titersbetween pGag and pTrimer-Gag-CD40L groups, with both groups reducingviral load by ˜1 log compared to pcDNA3.1 treated mice. In contrastpSPD-Gag-CD40L reduced mean viral load by ˜3 log in this experiment.

Mice Vaccinated with an Ad5-SPD-Gag-CD40L Viral Vector were CompletelyProtected from Vaccinia-Gag Challenge

While DNA vaccination is effective in mice, its translation to humanshas proved difficult. Instead, most currently tested HIV-1 vaccines haveused viral vectors, especially adenovirus-5 (Ad5). Consequently, thenucleic acid sequences for Gag alone (Ads-Gag) or SPD-Gag-CD40L(Ad5-SPD-Gag-CD40L) were cloned into replication defective Ad5 and usedto vaccinate mice twice at two-week intervals with 1×10E9 viralparticles (vp) i.m. Two weeks following the final vaccination, mice werechallenged intraperitoneally with vaccinia-Gag (107 PFU). Remarkably,all 5 mice vaccinated with Ad5-SPD-Gag-CD40L had no detectable vacciniavirus in their ovaries (<10 PFU/mouse) (FIG. 12), which wasstatistically significant compared with either the Ad5-Gag orunvaccinated groups (p<0.01). Overall there was a 7-log reduction invaccinia virus titers when Ad5-SPD-Gag-CD40L was compared to Ad5-Gag. Arepeat experiment gave similar results (data not shown). These datasupport the strategy of introducing SPD-Gag-CD40L into viral vectorvaccines such as Ad5.

Discussion

Stimulation through the CD40 receptor is important for generating CD8+ Tcell responses under non-inflammatory conditions. Numerous studies inmice have shown that agonistic antibodies to CD40 can activate strongresponses to vaccination. However, the translation of agonisticanti-CD40 antibody to the clinic has proved challenging due to concernsabout toxicity, depletion of CD40-positive cells such as B cells, andthe relatively limited effectiveness of agonistic anti-CD40 antibody inhumans when compared to studies in mice.

An important advance in the understanding of the CD40L/CD40 system hasbeen the recognition that DC activation requires clustering of the CD40receptor in order to stimulate the formation of an intracytoplasmicsignaling complex. For agonistic anti-CD40 antibodies, clusteringrequires that the antibodies be mounted via FcRs on an adjacent cell.Under conditions where an adjacent FcR-bearing cell is absent, agonisticanti-CD40 antibodies are not effective.

Keeping in mind this requirement for CD40 receptor clustering, we andothers have examined various multi-trimer forms of CD40L as agonists formurine, macaque, and human DCs. These molecules were made as fusionproteins between a multimerization scaffold such as SPD and theextracellular domain of CD40L. SPD is an ideal scaffold because CD40L isa Type II membrane protein in which the C-terminus faces outward and SPDforms a plus sign-shaped structure where the N-terminus is at thecentral “hub” and the C-terminus faces conveniently outward. When usedas a DNA vaccine, multi-trimer SPD-CD40L was an effective adjuvant whenadded to plasmid DNA encoding an antigen and led to significantlyincreased antigen-specific CD8+ T cell responses. However, wehypothesized that the vaccine response might be even stronger if theantigen and multi-trimer CD40L protein sequences were physically linkedrather than being mixed together for vaccination. Consequently, atripartite fusion protein was constructed that combined the SPDmultimerization scaffold, HIV-1 Gag as an antigen, and murine CD40L asthe adjuvant (SPD-Gag-CD40L) (FIGS. 5A and 5B).

As a first step, non-denaturing PAGE was used to show that SPD-Gag-CD40Lprotein is indeed a high molecular weight multimer complex (FIGS. 6C and6D). In vitro, this multi-trimer CD40L molecule could stimulate a CD40receptor-bearing indicator cell line that reports out NF-κB activationby releasing secreted alkaline phosphatase (SEAP) (FIG. 7A). As acontrol, a molecule was made in which the N-terminal “hub” of SPD wasdeleted, leading to a 1-trimer CD40L molecule that had little or noactivating in this NF-κB activation assay (data not shown). This controlrevealed the critical importance of the multi-trimer structure informing a highly actively form of CD40L, as previously demonstrated byHaswell et al. (Eur J Immunol. 2001; 31(10):3094-100). As expected,SPD-Gag-CD40L stimulated murine bone marrow-derived DCs in vitro toexpress cell surface markers of activation (FIG. 7B). While these datado not present direct evidence that the SPD-Gag-CD40L constructs foldsinto the structure outlined in FIG. 5B, we consider the ability of theconstruct to form biologically active trimers to provide initialevidence that functional trimers are being generated. In preliminaryexperiments we have also observed biological activity for SPD-CD40Lfusions with alternative antigens including gp100 and HIV-1 Env gp120(data not shown), supporting the concept that SPD-CD40L fusions withantigen is broadly applicable as a vaccine design strategy.

In vivo, plasmid DNA (pSPD-Gag-CD40L) was tested as a vaccine (FIG. 8A)and compared to vaccination with plasmid DNA for Gag alone (pGag) or anmixture of separate pGag antigen plasmid with pSPD-CD40L adjuvantplasmid. Strikingly, pSPD-Gag-CD40L elicited the strongest CD8+ T cellresponses as judged by the number of IFN-γ and IL-2 producing cells inan ELISPOT analysis (FIGS. 8B, 8C, 10A and 10B). pSPD-Gag-CD40L elicitedCD8+ T cells with remarkably increased avidity for the Gag peptideantigen (FIG. 9A). However, as we and others have previously described,multi-trimer CD40L is not a good adjuvant for antibody responses (FIG.9B), which emphasizes the special effects of CD40L on DCs and subsequentCD8+ T cell responses. While CD40L plays a role in promoting B-cellproliferation and immunoglobin class switching, several reports haveshown that strong CD40 stimulation can also prevent the movement of Bcells into germinal centers, block the development of memory B cells,and impair B-cell differentiation into antibody-secreting plasma cells.We have also observed similar responses by SPD-CD40L in previousstudies. We propose that SPD-Gag-CD40L is unable to enhance antibodyresponses through one or more of these mechanisms.

In addition, these CD8+ T cell responses were protective as judged bythe 2-3 log reduction in tissue viral load after challenging the micewith vaccinia-Gag (FIGS. 11A and 11B). However, we note that viraltiters following SPD-Gag-CD40L vaccination were not significantlydifferent than viral titers following vaccination with Gag plusSPD-CD40L, despite a large difference in interferon gamma and IL-2ELISPOT responses between the two groups. Partly this may reflect theinherent variability of DNA vaccine immune responses, given that 4/13mice given SPD-Gag-CD40L were able to clear virus while Gag plusSPD-CD40L was unable to reduce titer below 104 pfu/mouse. Overall, Gagplus SPD-CD40L gave a similar mean viral titer to Gag plus empty vector.Since DNA vaccination is a relatively inefficient way to deliver agenetic construct, an adenoviral vector (Ad5) was also used to vaccinatemice. Very remarkably, there was a ˜7 log reduction in tissue viral loadin mice vaccinated with Ad5-SPD-Gag-CD40L and no challenge virus couldbe detected (FIG. 12).

To account for the effectiveness of the SPD-Gag-CD40L vaccine design,three factors should be considered: (1) Use of multi-trimer CD40L tocluster the CD40 receptor and thereby activate DCs; (2) Role of CD40L intargeting antigen to CD40 receptor-bearing DCs; and (3) simultaneousdelivery of both the Gag antigen and CD40L adjuvant to the same DC atthe same time.

(1) Regarding the multi-trimer nature of CD40L in SPD-Gag-CD40L, it isworth noting that others have previously made antigen-CD40L fusionproteins. Xiang et al. (J. Immunol. 2001; 167(8):4560-5) fused a tumorantigen to the C-terminal end of CD40L in a position that couldconceivably impair binding of the ligand to the CD40 receptor. No datawere presented to rule out this concern, but the vaccine's effectivenesswas modest. Similarly, Zhang et al. fused a tumor antigen onto theN-terminus of the CD40L extracellular domain and delivered thisconstruct using an adenovirus vector. In this case, the molecular designallowed for CD40L to bind unimpaired to its receptor. Even so, theeffectiveness of this vaccine was relatively modest (Proc Natl Acad SciUSA. 2003; 100(25):15101-6). This is expected when a 1-trimer form ofCD40L is used rather than a receptor-clustering multi-trimer constructsuch as SPD-Gag-CD40L.

(2) Regarding the targeting of antigen to CD40 on DCs, this has emergedas a very desirable property for vaccine design. Barr et al. showed thatantigen conjugated to anti-CD40 antibody elicited strong vaccineresponses, although toxicity and anti-idiotypic antibody development aredrawbacks to this approach (Barr et al., Immunology. 2003;109(1):87-92). In vitro, Flamar et al. showed that anti-CD40 antibodyconjugated to five HIV antigenic peptides could be taken up by human DCsin vitro and the antigens were then presented to T cells from the bloodof HIV-infected subjects (Flamar et al., AIDS. 2013 Aug. 24;27(13):2041-51). In vivo, Cohn et al. found that conjugating antigen toanti-CD40 antibody broadened the types of DCs that crosspresent antigento T cells to include BDCA1(+) DCs in addition to standardcrosspresentation by BDCA3(+) DCs (J Exp Med. 2013; 210(5):1049-63.PMCID: 3646496). However, DC crosspresentation alone does not generateCD8+ T cell responses. As shown by Bonifaz and Steinman, antigenconjugated to anti-DEC205 antibody was targeted to DCs but theunactivated DCs lead to abortive T cell responses and subsequenttolerance. As they showed, the induction of CD8+ T cell responses by theanti-DEC205 antibody/antigen vaccine also required the addition of aDC-activating CD40 stimulus ((Bonifaz et al., J Exp Med. 2002;196(12):1627-38). Thus, targeting of antigen to CD40 is helpful but notsufficient for DC-mediated T cell activation and expansion. Indeed,targeting a vaccine antigen to unactivated DCs could becounterproductive and lead to tolerance rather than augmented vaccineresponses.

(3) Regarding the need for delivery of both antigen and adjuvant to thesame DC at the same time, this issue was recently examined by Kamath etal. (J Immunol. 2012; 188(10):4828-37). When antigen was delivered toDCs in the absence of adjuvant, antigen-specific T cells were induced toproliferate but did not subsequently differentiate into effector cells.Instead, effective immunity was only induced when the test vaccineprovided antigen and adjuvant to the same individual DCs within a shortwindow of time. These parameters are fulfilled by the design ofSPD-Gag-CD40L because the antigen and adjuvant are linked in time andspace as parts of the very same molecule.

In conclusion, a vaccine was developed that combines multi-trimer CD40Las an adjuvant covalently linked to HIV-1 Gag antigen. Extremely strongand highly protective CD8+ T cell responses were induced by thisvaccine, especially when the construct was incorporated into an Ad5vector. Since other antigens can be substituted for HIV-1 Gag inSPD-Gag-CD40L, this immunogen design suggests a general method forconstructing an effective preventative and/or therapeutic vaccine forinfections and tumors for which a strong CD8+ T cell response isrequired.

EXAMPLE 2 Cancer Immunotherapy

Previous studies have shown that plasmid DNA vaccination using anexogenous gene encoding tumor associated antigens can inducecancer-specific CTLs with antitumor activity. A second-generationimprovement on this approach is the targeting of antigen to dendriticcells (DC) by fusion to antibodies or natural ligands that binddendritic cell receptors. Recently it has been shown that targeting ofantigen to DC via CD40 is particularly effective at inducing crosspresentation of targeted antigens.

In this example we explored the use of CD40 ligand to target tumorantigen to DC. A DNA vaccine was generated encoding a single fusionprotein composed of the spontaneously multimerizing gene SurfactantProtein D (SPD), gp100 tumor antigen, and the extracellular domain ofCD40L. This “third generation” antigen-CD40L approach was developed toboth target antigen to DC and optimally activate dendritic cells byclustering CD40 on the cell membrane. SPD-gp100-CD40L was expressed as asingle 110 kDa protein strand that self-assembles inside cells into amolecule with four trimeric arms containing 4 trimers of CD40L. Theprotein was biologically active on dendritic cells and able to induceCD40-mediated signaling. SPD-gp100-CD40L was evaluated in a B16-F10melanoma DNA vaccine model either alone or in combination with plasmidsencoding IL-12p70 and GM-CSF. Vaccination withSPD-gp100-CD40L+IL-12p70+GM-CSF significantly increased survival andinhibited tumor growth compared to all other treatments. Expression ofgp100 and SPD-CD40L as separate molecules did not enhance survival,suggesting incorporation of gp100 within the SPD-CD40L polymer isrequired for activity. These data support a model where gp100 antigenincorporated within SPD-CD40L multi-trimers targets antigen to DC invivo, induces activation of these DC, increases cross-presentation ofgp100 antigen, and generates a protective anti-tumor T cell responsewhen given in combination with IL-12p70 and GM-CSF molecular adjuvants.

Cancer vaccination has attracted renewed attention as a therapy for thetreatment of tumor growth and metastasis. The use of Tumor AssociatedAntigens TAN is particularly promising. Therapeutic effects specific tocancer cells can be generated through the careful selection of TAApreferentially expressed on tumor cells. In particular, it has beenreported that DNA vaccination using an exogenous plasmid encoding a TAAcan induce cancer-specific cytotoxic T lymphocytes (CTL) with antitumoractivity. However, optimal CTL activity requires that the antigen beselectively and efficiently presented by antigen presenting cells (APC)such as dendritic cells (DC), which play a pivotal role in theinitiation, programming and regulation of cancer-specific immuneresponses. One strategy to enhance the effectiveness of DNA vaccinesencoding weakly immunogenic antigens is by co-delivering genes encodingmolecular adjuvants that stimulate DC. TNF superfamily ligands (TNFSF)are costimulatory molecules involved in DC and T cell activation andhave previously been tested as adjuvants to enhance immune responses inseveral vaccination studies, in particular the DC activating moleculeCD40L, the cognate ligand for CD40.

Melanoma-specific antigen gp100, encoded within DNA or viral vectorvaccines, is known to induce measurable immune responses and suppresstumor growth. However, molecular adjuvants could enhance the overallimmune response to this antigen, inducing an effective immune responseable to prevent tumor growth. As important, targeting of tumor antigensdirectly to DC using the DC receptor DEC-205 has previously been shownto increase immune responses. Similarly, it has also been shown thatdelivery of antigens to DC via CD40 can enhance cross-presentation ofantigen to CD8+ T cells via MHC I.

CD40L stimulation increases effector T cell differentiation and alsoinduces the production of a variety of cytokines, such as IL-12p70.Based on previously published data, a 4-trimer soluble form of CD40L hasbeen shown to be particularly effective as a vaccine adjuvant. This4-trimer soluble form was achieved using the scaffold protein SurfactantProtein D (SPD), a collectin family member that spontaneously forms aplus-sign-shaped molecule with four trimeric arms, generating a 4-trimersoluble complex.

In addition to CD40L, other adjuvants previously tested in cancervaccine models include GM-CSF and IL-12p70. Systemic co-administrationof IL-12p70 or GM-CSF have been shown to induce antitumor immunity.Studies have also evaluated these cytokines as DNA-encoded adjuvants forDNA vaccines where they have shown modest efficacy.

In the present study, the fusion protein SPD-gp100-CD40L was generatedencoding murine CD40L extracellular domain fused to the collagen-likedomain of murine SPD, with gp100 antigen inserted within the SPD codingregion. We reasoned that these soluble CD40L multi-trimers would delivergp100 to DC while simultaneously activating the DC, thereby inducing anenhanced CD8+ T cell CTL response. As we report, SPD-gp100-CD40L proteinwas stable, formed large polymeric complexes, and was biologicallyactive on DC, suggesting proper assembly of CD40L trimers. Co-deliveryof SPD-gp100-CD40L, GM-CSF, and IL-12p70 plasmids by intramuscularinjection enhanced survival of mice challenged with B16-F10 andsignificantly suppressed tumor growth. This response was not observedwith any other DNA vaccine combination, and was not observed when gp100and SPD-CD40L were delivered as separate molecules, either in presenceor absence of GM-CSF and IL-12p70. Overall, these data support thehypothesis that SPD-gp100-CD40L, when augmented with GM-CSF and IL-12p70cytokines, targets gp100 antigen to DC in situ, activates these DC viaCD40 stimulation, and induces an immune response that controls tumorgrowth and enhances survival.

Materials and Methods Construction and Preparation of DNA Plasmids

Plasmid encoding human glycoprotein 100 (pgp100) was a gift of Dr.Patrick Hwu. Plasmid encoding the 4-trimer soluble form of murineSPD-CD40L was generated as previously described (Stone et al., J Virol.2006; 80(4):1762-72). To construct pSPD-gp100-CD40L, DNA encoding aminoacids 25 to 596 (sequence KVPRNQD to EAGLGQV) of human gp100,incorporating the full extracellular domain or gp100, was insertedbetween amino acids 105 and 106 of mouse SPD within construct SPD-CD40L(i.e. between peptide sequences GERGLSG and PPGLPGI of murine SPD).Murine IL-12p70 plasmid pIL-12 was purchased from Invivogen and encodesa single chain dimer of IL-12 p35 and p40 (InvivoGen). Murine GM-CSFplasmid was constructed using a codon-optimized gene encoding murineGM-CSF inserted into plasmid pcDNA3.1. Clone pgp100-IRES-SPD-CD40L wasgenerated by placing an IRES sequence between human gp100 (amino acids1-594) and murine SPD-CD40L (Zhou et al., Proc Natl Acad Sci USA. 2008;105(14):5465-70). All plasmids were propagated in Escherichia colistrain TOP10. Highly purified, endotoxin-free DNA plasmid preparationswere produced using the Qiagen endofree GIGA plasmid kit. Plasmids werefurther purified using a Triton-X114 purification method as previouslydescribed (Stone et al., J Virol. 2006; 80(4):1762-72). All plasmidendotoxin levels were <0.2 EU/ml as confirmed by LAL endotoxin assay(Lonza Inc.).

Transient Transfections and Western blotting of Fusion ProteinConstructs

293T cells were transiently transfected with plasmid constructs usingGenjet Plus transfection reagent (Signagen Laboratories). Forty-eighthours later, supernatants were centrifuged and filtered. Supernatantswere loaded onto a sodium-dodecyl sulfate-10% polyacrylamide gel(BioRad) in the presence of DTT, electrophoresed, and blotted onto PVDFmembrane (Pierce). The membrane was blocked using 5% (w/v) dry milk andthen probed with goat anti-mouse CD40L antibody (R&D Systems), followedby incubation with anti-goat horseradish peroxidase-conjugatedantibodies (Jackson Immunoresearch). The protein band was developed ontoX-ray film using chemiluminescence. For analytical light scatteringanalysis, 293T cells were transiently transfected with thepSPD-gp100-CD40L construct and supernatant was collected and thenconcentrated 10-fold using an Amicon centrifugal filtration system with100 kDa cutoff (Millipore).

CD40 SEAP in Vitro Activity Assay

The CD40 receptor bearing reporter cell line CD40-293-SEAP was used tomonitor CD40L mediated activation. This 293-derived cell lineconstitutively expresses human CD40 receptor along with the gene forsecreted alkaline phosphatase (SEAP) under the control of NF-□B [59].Briefly, 80,000 CD40-293-SEAP reporter cells grown in DMEM medium with10% FBS were plated in each well of a 96-well plate. A total of 100 μlof SPD-gp100-CD40L, SPD-CD40L or pcDNA3.1 transfected 293T cellsupernatant was added to the cells in triplicate at various dilutions.After 18 hours, 10 μl/well of the supernatant from each well was addedto a 96-well assay plate together with 100 μl/well of QUANTI-BlueAlkaline Phosphatase substrate (InvivoGen). Wells were incubated for 20min at 20° C. and read at 650 nm in a 96-well plate reader.

DC Activation and Maturation Assay

Bone marrow derived DC were generated by standard methods with thefollowing modifications. Bone marrow cells were obtained from C57BL/6mice and washed in RPMI 1640 media. The cells were then placed in anon-tissue culture treated T75 flask at a concentration of 1×106 cellsper ml in 20 ml complete RPMI (RPMI 1640 with 10% FBS, 20 μg/mlgentamycin sulfate, 50 μM Mercaptoethanol), 20 ng/ml murine recombinantGM-CSF and 10 ng/ml murine recombinant IL-4 (Peprotech, Rocky Hill,N.J.)). Cells were cultured at 37° C., 5% CO2 and on day 3, media wasreplaced with fresh complete RPMI containing cytokines. On day 5, cellswere harvested, washed and resuspended in complete RPMI at 5×105cells/ml. A total of 1×106 cells were added to each well of 6-wellnon-tissue culture treated plates. Subsequently, 300 μl of supernatantcontaining SPD-gp100-CD40L, pcDNA3.1 control supernatant, or cytokinemix positive control (15 ng/ml IL-1beta, 5 ng/ml TNFalpha, and 1 μg/mlPGE2 final concentration) was added and cells were incubated for 36hours. Cells were harvested and stained with Hamster anti-mouse CD11cclone N418 PE-Cyanine7 conjugate (eBioscience, San Diego, Calif.)combined with each of the following antibodies: anti-mouse CD80 clone16-10A1, anti-mouse CD86 clone GL1, anti-mouse CD40 clone 1C10,anti-mouse CD83 clone Michel-17, anti-mouse MHC Class II (I-A/I-E)cloneM5-114.15.2, and anti-mouse CCR7 clone 4B12 (all from eBioscience).After flow cytometry analysis, the mean fluorescence intensity wascalculated for gated CD11c+dendritic cells under each experimentalcondition. FlowJo 7.6.4, flow cytometry analysis software, (FlowJo,Ashland, Oreg.) was used for analysis. Three independent wells wereanalyzed for each condition.

Tumor Immunotherapy Studies

Female C57BL/6 mice (7-8 weeks old) were used in all experiments.Animals were housed at the University of Miami under the guidelines ofthe National Institutes of Health (NIH, Bethesda, Md.). Animalexperiments were performed in accordance with national and institutionalguidance for animal care and were approved by the IACUC of theUniversity of Miami. A total of 50,000 B16-F10 cells were injected i.d.into the left flank. Mice were then injected i.m. with plasmid DNA onday 3, 10, and 17 following tumor challenge into both hind quadricepsmuscles. Mice received a mixture of from one to three plasmidconstructs. Empty vector pcDNA3.1 was used as filler to ensure allgroups received the same total micrograms of plasmid. Tumor volume wasmeasured 3 times per week using a digital caliper, measuring the longestdiameter (a) and shortest width (b) of the tumor. Tumor volume wascalculated by the formula V (mm3)=0.5×ab2. Animals were euthanized whentumors reached >1500 mm3. For GVAX vaccination, B16-F10 tumor cellsexpressing GM-CSF, kindly provided by Dr. Glenn Dranoff, were irradiated(5,000 rad) and 1×106 cells were injected subcutaneously on the rightflank on day 3, 6, and 9.

Histology

Tumors were harvested for histological analysis on day 15-20, fixing thetissue overnight at 4° C. in 10% formalin prior to embedding inparaffin. Serial 4 μm sections were then stained with hematoxylin andeosin (H&E) to evaluate for the presence of lymphocyte infiltration.

Statistical Analysis

Graph pad Prism 6.0 software was used to calculate significance bytwo-tailed Student's t test. In all figures, p values were labeled byasterisks for p<0.05 (*), p<0.01 (**), and p<0.001 (***).

Results Construction and Expression of Multi-Trimeric SolubleSPD-gp100-CD40L

Previous studies have shown that CD40L-mediated signaling is requiredfor functional CTL memory development against tumors. Similarly, we havepreviously shown that injection of plasmid DNA expressing SPD-CD40L intoB16-F10 tumors can slow tumor growth when combined with TLR agonists.CD40L mediates the co-stimulation, activation, and maturation ofdendritic cells (DC), and this function is critical for the induction ofan effective T cell mediated immune response. Previous research hasshown that monoclonal antibodies targeting DC surface protein DEC-205can target cancer antigens to DC in vivo, inducing a protective immuneresponse. We surmised that SPD-CD40L could similarly be used as acarrier to transport tumor associated antigens (TAA) to DC in vivo byincorporating the antigen within the SPD collagen-like domain ofSPD-CD40L. We constructed the plasmid pSPD-gp100-CD40L, where humangp100 is fused between amino acids 105 and 106 of the collagen-likedomain of murine SPD-CD40L (FIG. 13A) and SEQ ID NO 5 and SEQ ID NO 6. Amodel of the expected 4-trimer complex is shown in FIG. 13B. Followingtransfection of pSPD-gp100-CD40L into 293T cells, secretedSPD-gp100-CD40L was detected at the expected size of 110 KDa by SDS-PAGEWestern blot in the presence of DTT (FIG. 13C).

Biological Activity of SPD-gp100-CD40L

To confirm that SPD-gp100-CD40L retains biological activity, an SEAPcell line reporter assay was performed as described previously. Wemonitored the ability of SPD-gp100-CD40L supernatant to driveNF-kappaB-mediated expression of the SEAP reporter enzyme. Empty vectorpcDNA3.1 transfected 293T cell supernatant was used as a negativecontrol. As shown in FIG. 14A, both SPD-CD40L and SPD-gp100-CD40Linduced SEAP activity at a similar level in a dose-dependent manner whencompared to empty vector.

Next, we evaluated the ability of SPD-gp100-CD40L to activate bonemarrow derived DCs. DCs were cultured with supernatant from 293T cellstransfected with either empty vector pcDNA3.1 or pSPD-gp100-CD40L. Acytokine mix containing recombinant IL-1beta, TNFalpha, and PGE2 (Mimic)was used as a positive control. We observed a significant increase inCD80, CD86 and CD83 MFI (comparing pcDNA3.1 to pSPD-gp100-CD40Lsupernatant). SPD-gp100-CD40L was moderately active compared to theMimic positive control.

SPD-gp100-CD40L DNA Alone Did Not Inhibit B16-F10 Tumor Growth In Mice

We next investigated the anti-tumor efficacy of pSPD-gp100-CD40Lplasmid, using a B16-F10 melanoma therapeutic vaccination model (FIG.15). Mice were divided into three vaccination groups: (i) PBS, (ii)pSPD-gp100-CD40L, and (iii) GVAX therapy. Group (ii) received 100 μg ofpSPD-gp100-CD40L i.m. per vaccination. We did not observe a statisticaldifference in tumor sizes and survival between groups (FIGS. 15B and15C), suggesting that pSPD-gp100-CD40L alone is insufficient to inducean anti-tumor activity.

The Combination of pSPD-gp100-CD40L, pGM-CSF, and pIL-12p70 InhibitedTumor Growth and Enhanced Survival Following B16-F10 Tumor Challenge

Next, we investigated whether SPD-gp100-CD40L activity could be enhancedusing the molecular adjuvants GM-CSF and IL-12p70. We hypothesized thatDC chemoattraction induced by GM-CSF and T cell costimulation induced byIL-12p70 would synergize with the CD40L-mediated DC activation inducedby SPD-gp100-CD40L, increasing the overall anti-tumor immune response.Mice were divided into 5 vaccination groups: (i) PBS, (ii)pSPD-gp100-CD40L+pGM-CSF, (iii) pSPD-gp100-CD40L+pIL-12, (iv)pSPD-gp100-CD40L+pGM-CSF+pIL-12, and (v) GVAX. Empty vector pcDNA3.1 wasused as filler to ensure all DNA vaccine groups received the samequantity of total plasmid (120 μg). All DNA vaccinations contained 80 μgof pSPD-gp100-CD40L and 20 μg each of pGM-CSF, pIL-12, and/or pcDNA3.1.The mean tumor size for group (iv) (SPD-gp100-CD40L+GM-CSF+IL-12) wassignificantly lower compared to groups (i), (ii), and (iii) on days 15,17, and 20 (FIG. 16B). We observed a statistically significantdifference in survival between group (iv) and groups (i), (ii) and (iii)(P<0.05) (FIG. 16C), and a statistically significant difference intumor-free survival between group (iv) and groups (i), (ii), and (iii)(p<0.01). As shown in FIG. 16D, five out of five mice in group (iv) werefree of palpable tumors on day 11 while five out of five mice in groups(i) (ii) and (iii) had palpable tumors on day 11. GVAX “gold standard”vaccination slowed tumor growth compared to untreated animals, howeverneither tumor growth nor survival reached statistical significance whencomparing GVAX to other groups (FIGS. 16B and 16C).

Alternative Combinations of gp100, SPD-CD40L, IL-12, and GM-CSF Fail toControl of B16-F10 Tumor Growth

The previous experiments did not evaluate all possible combinations ofgp100, SPD-CD40L, GM-CSF, and IL-12. We therefore wished to confirm thatphysically linking gp100 and SPD-CD40L was required for activity. Sixgroups were evaluated: (i) PBS, (ii) pgp100, (iii) pgp100+pGM-CSF, (iv)pgp100+pIL-12, (v) pgp100+pGM-CSF+pIL-12, and (vi) pgp100-IRES-SPD-CD40L(gp100 and SPD-CD40L expressed as separate molecules)+pIL-12+pGM-CSF.Empty vector pcDNA3.1 was used as filler to ensure all DNA vaccinegroups received the same quantity of plasmid (120 μg total, including 80μg of the gp100-containing plasmid and 20 μg each of pGM-CSF, pIL-12,and/or pcDNA3.1). We observed no statistical difference in mean tumorsizes between any of the six groups (FIG. 17B). We also failed toobserve a statistical difference in survival between groups (FIG. 17C).

Discussion

Recent advances in cancer immunotherapy support the concept that theimmune system can induce effective antitumor responses. In this contextit has been reported that DNA vaccination is effective for theprevention of metastasis and relapse. In particular, the application ofDNA vaccination against melanoma has shown promise following theidentification of tumor associated antigens (TAA) including gp100,MART-1 and TRP2. For the most part, melanoma DNA therapeutic vaccinesare based on the expression of full length antigen followingintramuscular injection or electroporation of plasmid DNA. The antigenis secreted from the vaccination site and taken up by APC at the vaccinesite or the local draining lymph node. However, it is becomingrecognized in the field that targeting cancer antigens directly to APC(in particular dendritic cells) induces a more effective immune responsecompared to untargeted tumor antigens. We hypothesized that fusingmelanoma antigen gp100 within the SPD collagen-like domain of SPD-CD40Lmulti-trimeric clusters would: 1) target gp100 to DC expressing CD40 insitu, 2) induce cross presentation of gp100 by these DC, possibly viadelivery of gp100 to the early endosome, and 3) activate and mature theDC via CD40 crosslinking with CD40L multi-trimers on the DC membranesurface. The SPD-gp100-CD40L fusion protein is a single gene 3.1 kb insize that can be easily encoded within DNA, RNA, or viral vector cancervaccines. Initially, we determined that SPD-gp100-CD40L was efficientlysecreted from transfected cells and formed large multimeric complexes.Western blotting showed that SPD-gp100-CD40L was expressed and secretedinto the culture supernatant at the expected molecular weight of 110kDa. We also confirmed the biological activity of SPD-gp100-CD40Lprotein using an NF-κB reporter system and DC activation assay. Togetherthese data suggest that SPD-gp100-CD40L is forming a biologically activetrimeric CD40L headgroup, in a manner similar to the previouslycharacterized SPD-CD40L protein, and these trimers are formingspontaneous 4-trimer complexes, consistent with the native SPD protein.

In a cancer model, therapeutic immunization with SPD-gp100-CD40L DNAvaccine failed to control tumor growth or improve survival of B16-F10melanoma (FIG. 16). This is not surprising, given the aggressive natureof established B16-F10 tumor. One possibility is that secretion ofimmunosuppressive cytokines such as VEGF, IL-10 and TGF-B by B16-F10prevents activated cytotoxic T lymphocytes (CTL) induced bySPD-gp100-CD40L from entering into the tumor bed. Alternately, these andother immunosuppressive cytokines suppress cytotoxic activity once theCTL enters the tumor tissue. Previous studies have evaluated cytokinesIL-12 and GM-CSF for their ability to enhance T cell mediated immuneresponses. We hypothesized that SPD-gp100-CD40L combined with cytokinesIL-12 and GM-CSF would enhance antigen cross-presentation (viaSPD-gp100-CD40L) and immune activation (via GM-CSF and IL-12),overcoming tumor-mediated immune suppression. Consistent with thishypothesis, we observed that vaccination with all 3 genes significantlyslowed tumor growth, delayed tumor onset, and improved mouse survival(FIG. 17). Only the triple combination was effective, and all othercombinations failed to significantly suppress tumor growth or enhancesurvival (FIG. 16), including separate expression of gp100 and SPD-CD40L(together with IL-12 and GM-CSF). All animals received the same amountof plasmid (120 kg), allowing us to control for immune stimulationprovided by plasmid DNA itself. Based on the literature and our data wepropose a model where the effectiveness of SPD-gp100-CD40L is due to thetargeting of gp100 to DC, enhanced cross-presentation throughCD40-mediated delivery to the early endosome, and the capacity of CD40Lmulti-trimers to enhance DC activation and maturation. In this model,SPD-gp100-CD40L-mediated DC cross-presentation and activation, coupledwith IL-12-p70-mediated T cell stimulation and GM-CSF-mediatedchemoattraction of DC, generated an enhanced CD8+ T cell response thatwas able to overcome immune tolerance at the tumor site. Our resultsalso suggest that CD40L stimulation is a critical component of thisvaccine. We did not observe any reduction in tumor growth kinetics whengp100 alone was combined with IL-12 and GM-CSF, despite higher levels ofgp100 protein expression in pgp100 transfected cells compared topSPD-gp100-CD40L transfected cells (FIG. 13C). In addition, the separatedelivery of gp100 and SPD-CD40L molecules (using an IRES construct) wasunable to replicate the effect of SPD-gp100-CD40L (FIG. 17), consistentwith the requirement that gp100 be physically linked to the CD40Lmulti-trimers for optimal activity. Additional research will be requiredto determine whether multi-trimerization of CD40L plays a role in theactivity of this construct. Of interest, recent studies have shown thatdelivery of antigen via CD40 can enhance cross presentation to DC. Bothenhanced cross-presentation and the simultaneous antigen delivery and DCactivation to the same cell may explain the ability of SPD-gp100-CD40Lto induce a robust anti-tumor immune response.

In conclusion, this study demonstrates that the fusion of gp100 withinSPD-CD40L multi-trimers induces a response against B16-F10 melanoma whencombined with IL-12p70 and GM-CSF molecular adjuvants. Overall,SPD-gp100-CD40L is a novel cancer DNA vaccine reagent that providesCD40-mediated APC activation in the context of efficient targeting andcross-presentation of cancer antigen. Future studies will explorealternative SPD-TAA-CD40L fusion proteins using tumor-associatedantigens other than gp100. This will allow us to determine whether thisstrategy can be expanded to a wider range of cancers and TAA. Insummary, this study presents a novel reagent for use in cancertherapeutic vaccines, exploiting the unique properties of CD40L on theactivation of DC and using CD40L for the targeting and enhanced crosspresentation of antigen on APC.

SEQUENCE EXAMPLES

The following sequences further describe certain embodiments of theinvention:

<160> NUMBER OF SEQ ID NOS: 18 <210> SEQ ID NO 1 <211> LENGTH: 2913<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to HIV-1 Gag and the extracellular domain ofmurine CD40L muSP-D-GAG-muSP-D-muCD40LItalicized/bolded sequence: Murine SP-D sequence (collagen-like domain)Non-italicized/bolded sequence: HIV-1 Gag sequenceItalicized sequence: Murine CD40L sequence (extracellular domain)

ATGGGAGCCAGGGCCAGCGTGCTGTCTGGGGGCGAGCTGGACAGGTGGGAGAAGATTAGGCTGAGGCCCGGAGGAAAGAAGAAGTACAAACTGAAACACATCGTGTGGGCCTCCCGGGAGCTGGAACGGTTCGCCGTGAATCCTGGGCTGCTGGAGACCTCTGAGGGCTGCAGACAGATCCTGGGACAGCTGCAGCCTAGCCTGCAGACCGGAAGCGAGGAGCTGAGGTCTCTGTACAACACCGTGGCCACACTGTACTGCGTGCACCAGCGGATTGAGGTGAAGGATACCAAGGAAGCCCTGGAGAAGATTGAGGAAGAGCAGAATAAGTCCAAGAAGAAAGCCCAGCAGGCCGCCGCCGACACAGGAAATAGCTCCCAGGTGTCTCAGAACTACCCCATCGTGCAGAACCTGCAGGGACAGATGGTGCACCAGGCCATCAGCCCCCGGACCCTGAACGCCTGGGTGAAGGTGGTGGAAGAGAAAGCCTTCAGCCCAGAAGTGATCCCCATGTTCAGCGCCCTGAGCGAAGGGGCCACCCCACAGGACCTGAATACAATGCTGAATACAGTGGGCGGCCACCAGGCCGCCATGCAGATGCTGAAGGAGACCATTAACGAGGAGGCCGCCGAGTGGGATAGGCTGCACCCAGTGCACGCCGGGCCCATCGCCCCAGGGCAGATGAGGGAGCCACGGGGCTCTGACATCGCCGGCACCACCTCTACCCTGCAGGAGCAGATCGGCTGGATGACCAATAACCCACCTATTCCCGTGGGAGAAATCTACAAAAGGTGGATTATCCTGGGGCTGAACAAGATCGTGAGAATGTACTCCCCAACATCCATTCTGGACATCCGGCAGGGCCCAAAGGAACCCTTTAGAGACTACGTGGATAGGTTCTACAAAACCCTGCGCGCCGAGCAGGCCTCCCAGGAGGTGAAGAACTGGATGACCGAGACACTGCTGGTGCAGAATGCCAACCCAGACTGTAAGACCATTCTGAAGGCCCTGGGACCAGCCGCCACCCTGGAGGAAATGATGACAGCCTGCCAGGGGGTGGGCGGACCCGGCCACAAGGCCCGCGTGCTGGCCGAGGCCATGTCCCAGGTGACAAATTCCGCCACCATCATGATGCAGCGCGGAAATTTTCGGAATCAGCGCAAAACAGTGAAATGCTTCAATTGCGGGAAGGAGGGCCACATCGCCAAGAATTGCAGAGCCCCAAGGAAGAAGGGCTGCTGGAAGTGCGGAAAGGAGGGCCACCAGATGAAGGACTGCACAGAGCGCCAGGCCAATTTCCTGGGCAAGATCTGGCCATCCCACAAGGGGCGGCCTGGAAACTTCCTGCAGAGCCGGCCCGAACCCACAGCCCCCCCTGAAGAATCCTTCCGGTTCGGAGAGGAAACAACCACACCCAGCCAGAAGCAGGAGCCTATCGACAAGGAACTGTACCCACTGGCCAGCCTGAGAAGCCTGTTCGGCAACGATCCAAGCAGCCAG

CATAGAAGATTGGATAAGGTCGAAGAGGAAGTAAACCTTCATGAAGATTTTGTATTCATAAAAAAGCTAAAGAGATGCAACAAAGGAGAAGGATCTTTATCCTTGCTGAACTGTGAGGAGATGAGAAGGCAATTTGAAGACCTTGTCAAGGATATAACGTTAAACAAAGAAGAGAAAAAAGAAAACAGCTTTGAAATGCAAAGAGGTGATGAGGATCCTCAAATTGCAGCACACGTTGTAAGCGAAGCCAACAGTAATGCAGCATCCGTTCTACAGTGGGCCAAGAAAGGATATTATACCATGAAAAGCAACTTGGTAATGCTTGAAAATGGGAAACAGCTGACGGTTAAAAGAGAAGGACTCTATTATGTCTACACTCAAGTCACCTTCTGCTCTAATCGGGAGCCTTCGAGTCAACGCCCATTCATCGTCGGCCTCTGGCTGAAGCCCAGCATTGGATCTGAGAGAATCTTACTCAAGGCGGCAAATACCCACAGTTCCTCCCAGCTTTGCGAGCAGCAGTCTGTTCACTTGGGCGGAGTGTTTGAATTACAAGCTGGTGCTTCTGTGTTTGTCAACGTGACTGAAGCAAGCCAAGTGATCCACAGAGTTGGCTTCTCATCTTTTGGCTTACTCAAACTCTGA <210> SEQ ID NO 2<211> LENGTH: 970 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223>OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to HIV-1 Gag and the extracellular domain ofmurine CD40LMLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDGRDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERGLSGMGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEVKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGNDPSSQPPGLPGIPGPAGKEGPSGKQGNIGPQGKPGPKGEAGPKGEVGAPGMQGSTGAKGSTGPKGERGAPGVQGAPGNAGAAGPAGPAGPQGAPGSRGPPGLKGDRGVPGDRGIKGESGLPDSAALRQQMEALKGKLQRLEVAFSHYQKAALFPDGHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSIGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLtpa-muACRP30-gp120-muACRP30-muBAFF <210> SEQ ID NO 3 <211> LENGTH: 2784<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Human TPA signal sequence fused tomurine ACRP30 fused to HIV-1 Env gp120 and the extracellulardomain of murine BAFF Underlined sequence: Human TPA sequenceItalicized/bolded sequence: Murine ACRP30 sequenceNon-italicized/bolded sequence: HIV-1 Env gp120 sequenceItalicized sequence: Murine BAFF sequence (extracellular domain)ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCC CAGC

TGGGGCAACCTGTGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCAAGACCACCCTGTTCTGCGCCAGCGACGCCAAGAGCTACGAGAAGGAGGTGCACAACGTGTGGGCCACCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATCGTGCTGGGCAACGTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTGGACCAGATGCACGAGGACATCATCAGCCTGTGGGACCAGAGCCTGAAGCCCTGCGTGAAGCTGACCCCCCTGTGCGTGACCCTGAACTGCACCGAGGTGAACGTGACCCGCAACGTGAACAACAGCGTGGTGAACAACACCACCAACGTGAACAACAGCATGAACGGCGACATGAAGAACTGCAGCTTCAACATCACCACCGAGCTGAAGGACAAGAAGAAGAACGTGTACGCCCTGTTCTACAAGCTGGACATCGTGAGCCTGAACGAGACCGACGACAGCGAGACCGGCAACAGCAGCAAGTACTACCGCCTGATCAACTGCAACACCAGCGCCCTGACCCAGGCCTGCCCCAAGGTGAGCTTCGACCCCATCCCCATCCACTACTGCGCCCCCGCCGGCTACGCCATCCTGAAGTGCAACAACAAGACCTTCAACGGCACCGGCCCCTGCCACAACGTGAGCACCGTGCAGTGCACCCACGGCATCAAGCCCGTGGTGAGCACCCAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGGCATCATCATCCGCAGCGAGAACCTGACCAACAACGTCAAGACCATCATCGTGCACCTGAACCGCAGCATCGAGATCGTGTGCGTGCGCCCCAACAACAACACCCGCCAGAGCATCCGCATCGGCCCCGGCCAGACCTTCTACGCCACCGGCGACATCATCGGCGACATCCGCCAGGCCCACTGCAACATCAGCCGCACCAACTGGACCAAGACCCTGCGCGAGGTGCGCAACAAGCTGCGCGAGCACTTCCCCAACAAGAACATCACCTTCAAGCCCAGCAGCGGCGGCGACCTGGAGATCACCACCCACAGCTTCAACTGCCGCGGCGAGTTCTTCTACTGCAACACCAGCGGCCTGTTCAGCATCAACTACACCGAGAACAACACCGACGGCACCCCCATCACCCTGCCCTGCCGCATCCGCCAGATCATCAACATGTGGCAGGAGGTGGGCCGCGCCATGTACGCCCCCCCCATCGAGGGCAACATCGCCTGCAAGAGCGACATCACCGGCCTGCTGCTGGTGCGCGACGGCGGCAGCACCAACGACAGCACCAACAACAACACCGAGATCTTCCGCCCCGCCGGCGGCGACATGCGCGACAACTGGCGCAGCGAGCTGTACAAGTACAAGGTGGTGGAGATCAAGCCCCTGGGCATCGCCCCCACCGAGGCCAAGCGCCGCGTGGTGGAGCGCGAGAAGCGCGCCGTGGGCATCGGCGCCGTGTTCCTGGGCTTCCTGGGCGCCGCCGGCAGCACCATGGGCGCCGCCAGCATCACCCTGACCGCCCAGGCCCGCCAGGTGCTGAGCGGCATCGTGCAGCAGCAGAGCAACCTGCTGCGCGCCATCGAGGCCCAGCAGCACCTGCTGCAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGACCCGCGTGCTGGCCATCGAGCGCTACCTGAAGGACCAGCAGCTGCTG

CAGTTGGCTGCCTTGCAAGCAGACCTGATGAACCTGCGCATGGAGCTGCAGAGCTACCGAGGTTCAGCAACACCAGCCGCCGCGGGTGCTCCAGAGTTGACCGCTGGAGTCAAACTCCTGACACCGGCAGCTCCTCGACCCCACAACTCCAGCCGCGGCCACAGGAACAGACGCGCTTTCCAGGGACCAGAGGAAACAGAACAAGATGTAGACCTCTCAGCTCCTCCTGCACCATGCCTGCCTGGATGCCGCCATTCTCAACATGATGATAATGGAATGAACCTCAGAAACATCATTCAAGACTGTCTGCAGCTGATTGCAGACAGCGACACGCCGACTATACGAAAAGGAACTTACACATTTGTTCCATGGCTTCTCAGCTTTAAAAGAGGAAATGCCTTGGAGGAGAAAGAGAACAAAATAGTGGTGAGGCAAACAGGCTATTTCTTCATCTACAGCCAGGTTCTATACACGGACCCCATCTTTGCTATGGGTCATGTCATCCAGAGGAAGAAAGTACACGTCTTTGGGGACGAGCTGAGCCTGGTGACCCTGTTCCGATGTATTCAGAATATGCCCAAAACACTGCCCAACAATTCCTGCTACTCGGCTGGCATCGCGAGGCTGGAAGAAGGAGATGAGATTCAGCTTGCAATTCCTCGGGAGAATGCACAGATTTCACGCAACGGAGACGACACCTTCTTTGGTGCCCTAAAACTGCTGTAA <210> SEQ ID NO 4<211> LENGTH: 927 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223>OTHER INFORMATION: Human TPA signal sequence fused tomurine ACRP30 fused to HIV-1 Env gp120 and the extracellular domain of murine BAFFMDAMKRGLCCVLLLCGAVFVSPSEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHNGTPGRDGWGNLWVTVYYGVPVWKEAKTTLFCASDAKSYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTEVNVTRNVNNSVVNNTTNVNNSMNGDMKNCSFNITTELKDKKKNVYALFYKLDIVSLNETDDSETGNSSKYYRLINCNTSALTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEEGIIIRSENLTNNVKTIIVHLNRSIEIVCVRPNNNTRQSIRIGPGQTFYATGDIIGDIRQAHCNISRTNWTKTLREVRNKLREHFPNKNITFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFSINYTENNTDGTPITLPCRIRQIINMWQEVGRAMYAPPIEGNIACKSDITGLLLVRDGGSTNDSTNNNTEIFRPAGGDMRDNWRSELYKYKVVEIKPLGIAPTEAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTAQARQVLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLKDQQLLRDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEPGEAAQLAALQADLMNLRMELQSYRGSATPAAAGAPELTAGVKLLTPAAPRPHNSSRGHRNRRAFQGPEETEQDVDLSAPPAPCLPGCRHSQHDDNGMNLRNIIQDCLQLIADSDTPTIRKGTYTFVPWLLSFKRGNALEEKENKIVVRQTGYFFIYSQVLYTDPIFAMGHVIQRKKVHVFGDELSLVTLFRCIQNMPKTLPNNSCYSAGIARLEEGDEIQLAIPRENAQISRNGDDTFFGALKLL muSP-D-gp100-muSP-D-muCD40L <210> SEQ ID NO 5 <211>LENGTH: 3129 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to human gp100 and the extracellular domain of murine CD40LItalicized/bolded sequence: Murine SP-D sequence (collagen- like domain)Non-italicized/bolded sequence: Human gp100 sequenceItalicized sequence: Murine CD40L sequence (extracellular domain)

AAAGTACCCAGAAACCAGGACTGGCTTGGTGTCTCAAGGCAACTCAGAACCAAAGCCTGGAACAGGCAGCTGTATCCAGAGTGGACAGAAGCCCAGAGACTTGACTGCTGGAGAGGTGGTCAAGTGTCCCTCAAGGTCAGTAATGATGGGCCTACACTGATTGGTGCAAATGCCTCCTTCTCTATTGCCTTGAACTTCCCTGGAAGCCAAAAGGTATTGCCAGATGGGCAGGTTATCTGGGTCAACAATACCATCATCAATGGGAGCCAGGTGTGGGGAGGACAGCCAGTGTATCCCCAGGAAACTGACGATGCCTGCATCTTCCCTGATGGTGGACCTTGCCCATCTGGCTCTTGGTCTCAGAAGAGAAGCTTTGTTTATGTCTGGAAGACCTGGGGCCAATACTGGCAAGTTCTAGGGGGCCCAGTGTCTGGGCTGAGCATTGGGACAGGCAGGGCAATGCTGGGCACACACACCATGGAAGTGACTGTCTACCATCGCCGGGGATCCCGGAGCTATGTGCCTCTTGCTCATTCCAGCTCAGCCTTCACCATTACTGACCAGGTGCCTTTCTCCGTGAGCGTGTCCCAGTTGCGGGCCTTGGATGGAGGGAACAAGCACTTCCTGAGAAATCAGCCTCTGACCTTTGCCCTCCAGCTCCATGACCCCAGTGGCTATCTGGCTGAAGCTGACCTCTCCTACACCTGGGACTTTGGAGACAGTAGTGGAACCCTGATCTCTCGGGCACTTGTGGTCACTCATACTTACCTGGAGCCTGGCCCAGTCACTGCCCAGGTGGTCCTGCAGGCTGCCATTCCTCTCACCTCCTGTGGCTCCTCCCCAGTTCCAGGCACCACAGATGGGCACAGGCCAACTGCAGAGGCCCCTAACACCACAGCTGGCCAAGTGCCTACTACAGAAGTTGTGGGTACTACACCTGGTCAGGCGCCAACTGCAGAGCCCTCTGGAACCACATCTGTGCAGGTGCCAACCACTGAAGTCATAAGCACTGCACCTGTGCAGATGCCAACTGCAGAGAGCACAGGTATGACACCTGAGAAGGTGCCAGTTTCAGAGGTCATGGGTACCACACTGGCAGAGATGTCAACTCCAGAGGCTACAGGTATGACACCTGCAGAGGTATCAATTGTGGTGCTTTCTGGAACCACAGCTGCACAGGTAACAACTACAGAGTGGGTGGAGACCACAGCTAGAGAGCTACCTATCCCTGAGCCTGAAGGTCCAGATGCCAGCTCAATCATGTCTACGGAAAGTATTACAGGTTCCCTGGGCCCCCTGCTGGATGGTACAGCCACCTTAAGGCTGGTGAAGAGACAAGTCCCCCTGGATTGTGTTCTGTATCGATATGGTTCCTTTTCCGTCACCCTGGACATTGTCCAGGGTATTGAAAGTGCCGAGATCCTGCAGGCTGTGCCGTCCGGTGAGGGGGATGCATTTGAGCTGACTGTGTCCTGCCAAGGCGGGCTGCCCAAGGAAGCCTGCATGGAGATCTCATCGCCAGGGTGCCAGCCCCCTGCCCAGCGGCTGTGCCAGCCTGTGCTACCCAGCCCAGCCTGCCAGCTGGTTCTGCACCAGATACTGAAGGGTGGCTCGGGGACATACTGCCTCAATGTGTCTCTGGCTGATACCAACAGCCTGGCAGTGGTCAGCACCCAGCTTATCATGCCTGGTCAAGAAGCAGGCCTTGGGCAGGTT

CATAGAAGATTGGATAAGGTCGAAGAGGAAGTAAACCTTCATGAAGATTTTGTATTCATAAAAAAGCTAAAGAGATGCAACAAAGGAGAAGGATCTTTATCCTTGCTGAACTGTGAGGAGATGAGAAGGCAATTTGAAGACCTTGTCAAGGATATAACGTTAAACAAAGAAGAGAAAAAAGAAAACAGCTTTGAAATGCAAAGAGGTGATGAGGATCCTCAAATTGCAGCACACGTTGTAAGCGAAGCCAACAGTAATGCAGCATCCGTTCTACAGTGGGCCAAGAAAGGATATTATACCATGAAAAGCAACTTGGTAATGCTTGAAAATGGGAAACAGCTGACGGTTAAAAGAGAAGGACTCTATTATGTCTACACTCAAGTCACCTTCTGCTCTAATCGGGAGCCTTCGAGTCAACGCCCATTCATCGTCGGCCTCTGGCTGAAGCCCAGCATTGGATCTGAGAGAATCTTACTCAAGGCGGCAAATACCCACAGTTCCTCCCAGCTTTGCGAGCAGCAGTCTGTTCACTTGGGCGGAGTGTTTGAATTACAAGCTGGTGCTTCTGTGTTTGTCAACGTGACTGAAGCAAGCCAAGTGATCCACAGAGTTGGCTTCTCATCTTTTGGCTTACTCAAACTCTGA <210> SEQ ID NO 6 <211> LENGTH: 1042 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to human gp100 and the extracellular domainof murine CD40LMLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDGRDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERGLSGKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPPGLPGIPGPAGKEGPSGKQGNIGPQGKPGPKGEAGPKGEVGAPGMQGSTGAKGSTGPKGERGAPGVQGAPGNAGAAGPAGPAGPQGAPGSRGPPGLKGDRGVPGDRGIKGESGLPDSAALRQQMEALKGKLQRLEVAFSHYQKAALFPDGHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSIGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVEVNVTEASQVIHRVGESSFGLL KLtpa-hulgG1Fc-gp120-GCN4-huAPRIL <210> SEQ ID NO 7 <211> LENGTH: 3282<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Human tpa signal sequence fused tohuman IgG1 Fc region fused to HIV-1 Env gp120 fused to GCN4trimerization motif fused to the extracellular domain of human APRILUnderlined sequence: Human tpa signal sequenceItalicized/bolded sequence: Human IgG1 Fc domain to hinge regionNon-italicized/bolded sequence: HIV-1 Env gp120 sequenceBold sequence: GCN4 trimerization motifItalicized sequence: Murine APRIL sequence (extracellular domain)ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCC CAGC

TGGGGCAACCTGTGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCAAGACCACCCTGTTCTGCGCCAGCGACGCCAAGAGCTACGAGAAGGAGGTGCACAACGTGTGGGCCACCCACGCCTGCGTGCCCACCGACCCCAACCCCCAGGAGATCGTGCTGGGCAACGTGACCGAGAACTTCAACATGTGGAAGAACGACATGGTGGACCAGATGCACGAGGACATCATCAGCCTGTGGGACCAGAGCCTGAAGCCCTGCGTGAAGCTGACCCCCCTGTGCGTGACCCTGAACTGCACCGAGGTGAACGTGACCCGCAACGTGAACAACAGCGTGGTGAACAACACCACCAACGTGAACAACAGCATGAACGGCGACATGAAGAACTGCAGCTTCAACATCACCACCGAGCTGAAGGACAAGAAGAAGAACGTGTACGCCCTGTTCTACAAGCTGGACATCGTGAGCCTGAACGAGACCGACGACAGCGAGACCGGCAACAGCAGCAAGTACTACCGCCTGATCAACTGCAACACCAGCGCCCTGACCCAGGCCTGCCCCAAGGTGAGCTTCGACCCCATCCCCATCCACTACTGCGCCCCCGCCGGCTACGCCATCCTGAAGTGCAACAACAAGACCTTCAACGGCACCGGCCCCTGCCACAACGTGAGCACCGTGCAGTGCACCCACGGCATCAAGCCCGTGGTGAGCACCCAGCTGCTGCTGAACGGCAGCCTGGCCGAGGAGGGCATCATCATCCGCAGCGAGAACCTGACCAACAACGTCAAGACCATCATCGTGCACCTGAACCGCAGCATCGAGATCGTGTGCGTGCGCCCCAACAACAACACCCGCCAGAGCATCCGCATCGGCCCCGGCCAGACCTTCTACGCCACCGGCGACATCATCGGCGACATCCGCCAGGCCCACTGCAACATCAGCCGCACCAACTGGACCAAGACCCTGCGCGAGGTGCGCAACAAGCTGCGCGAGCACTTCCCCAACAAGAACATCACCTTCAAGCCCAGCAGCGGCGGCGACCTGGAGATCACCACCCACAGCTTCAACTGCCGCGGCGAGTTCTTCTACTGCAACACCAGCGGCCTGTTCAGCATCAACTACACCGAGAACAACACCGACGGCACCCCCATCACCCTGCCCTGCCGCATCCGCCAGATCATCAACATGTGGCAGGAGGTGGGCCGCGCCATGTACGCCCCCCCCATCGAGGGCAACATCGCCTGCAAGAGCGACATCACCGGCCTGCTGCTGGTGCGCGACGGCGGCAGCACCAACGACAGCACCAACAACAACACCGAGATCTTCCGCCCCGCCGGCGGCGACATGCGCGACAACTGGCGCAGCGAGCTGTACAAGTACAAGGTGGTGGAGATCAAGCCCCTGGGCATCGCCCCCACCGAGGCCAAGCGCCGCGTGGTGGAGCGCGAGAAGCGCGCCGTGGGCATCGGCGCCGTGTTCCTGGGCTTCCTGGGCGCCGCCGGCAGCACCATGGGCGCCGCCAGCATCACCCTGACCGCCCAGGCCCGCCAGGTGCTGAGCGGCATCGTGCAGCAGCAGAGCAACCTGCTGCGCGCCATCGAGGCCCAGCAGCACCTGCTGCAGCTGACCGTGTGGGGCATCAAGCAGCTGCAGACCCGCGTGCTGGCCATCGAGCGCTACCTGAAGGACCAGCAGCTGCTGATGAAACAGATCGAGGATAAGATTGAGGAAATCCTGAGCAAGATCTACCATATCGAGAACGAAATTGCTAGGATCAAAAAGCTGATCGGCGAGGTG ATGCCAGCCTCATCTCCAGGCCACATGGGGGGCTCAGTCAGAGAGCCAGCCCTTTCGGTTGCTCTTTGGTTGAGTTGGGGGGCAGTTCTGGGGGCTGTGACTTGTGCTGTCGCACTACTGATCCAACAGACAGAGCTGCAAAGCCTAAGGCGGGAGGTGAGCCGGCTGCAGCGGAGTGGAGGGCCTTCCCAGAAGCAGGGAGAGCGCCCATGGCAGAGCCTCTGGGAGCAGAGTCCTGATGTCCTGGAAGCCTGGAAGGATGGGGCGAAATCTCGGAGAAGGAGAGCAGTACTCACCCAGAAGCACAAGAAGAAGCACTCAGTCCTGCATCTTGTTCCAGTTAACATTACCTCCAAGGACTCTGACGTGACAGAGGTGATGTGGCAACCAGTACTTAGGCGTGGGAGAGGCCTGGAGGCCCAGGGAGACATTGTACGAGTCTGGGACACTGGAATTTATCTGCTCTATAGTCAGGTCCTGTTTCATGATGTGACTTTCACAATGGGTCAGGTGGTATCTCGGGAAGGACAAGGGAGAAGAGAAACTCTATTCCGATGTATCAGAAGTATGCCTTCTGATCCTGACCGTGCCTACAATAGCTGCTACAGTGCAGGTGTCTTTCATTTACATCAAGGGGATATTATCACTGTCAAAATTCCACGGGCAAACGCAAAACTTAGCCTTTCTCCGCATGGAACATTCCTGGGGTTTGTGAAACTATGA <210> SEQ ID NO 8 <211> LENGTH: 1093<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Human tpa signal sequence fused tohuman IgG1 Fc region fused to HIV-1 Env gp120 fused to GCN4trimerization motif fused to the extracellular domain of human APRILMDAMKRGLCCVLLLCGAVFVSPSSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKWGNLWVTVYYGVPVWKEAKTTLFCASDAKSYEKEVHNVWATHACVPTDPNPQEIVLGNVTENFNMWKNDMVDQMHEDIISLWDQSLKPCVKLTPLCVTLNCTEVNVTRNVNNSVVNNTTNVNNSMNGDMKNCSFNITTELKDKKKNVYALFYKLDIVSLNETDDSETGNSSKYYRLINCNTSALTQACPKVSFDPIPIHYCAPAGYAILKCNNKTFNGTGPCHNVSTVQCTHGIKPVVSTQLLLNGSLAEEGIIIRSENLTNNVKTIIVHLNRSIEIVCVRPNNNTRQSIRIGPGQTFYATGDIIGDIRQAHCNISRTNWTKTLREVRNKLREHFPNKNITFKPSSGGDLEITTHSFNCRGEFFYCNTSGLFSINYTENNTDGTPITLPCRIRQIINMWQEVGRAMYAPPIEGNIACKSDITGLLLVRDGGSTNDSTNNNTEIFRPAGGDMRDNWRSELYKYKVVEIKPLGIAPTEAKRRVVEREKRAVGIGAVFLGFLGAAGSTMGAASITLTAQARQVLSGIVQQQSNLLRAIEAQQHLLQLTVWGIKQLQTRVLAIERYLKDQQLLMKQIEDKIEEILSKIYHIENEIARIKKLIGEVMPASSPGHMGGSVREPALSVALWLSWGAVLGAVTCAVALLIQQTELQSLRREVSRLQRSGGPSQKQGERPWQSLWEQSPDVLEAWKDGAKSRRRRAVLTQKHKKKHSVLHLVPVNITSKDSDVTEVMWQPVLRRGRGLEAQGDIVRVWDTGIYLLYSQVLFHDVTFTMGQVVSREGQGRRETLFRCIRSMPSDPDRAYNSCYSAGVFHLHQGDIITVKIPRANAKLSLSPHGTFLGFVKLhuSP-D-NP-huSP-D-huCD40L-NST <210> SEQ ID NO 9 <211> LENGTH: 2769 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Human Surfactant Protein Dcollagen-like domain fused to H. influenza NP proteinand the extracellular domain of human CD40L (minusstalk region (Non-Stalk (NST)))Italicized/bolded sequence: Human SP-D sequence (collagen-like domain)Non-italicized/bolded sequence: H. influenza NP sequenceItalicized sequence: Human CD40L sequence (extracellulardomain, missing stalk region)

ATGGCGTCTCAAGGCACCAAACGATCTTACGAACAGATGGAGACTGATGGAGAACGCCAGAATGCCACTGAAATCAGAGCATCCGTCGGAAAAATGATTGGTGGAATTGGACGATTCTACATCCAAATGTGCACCGAACTCAAACTCAGTGATTATGAGGGACGGTTGATCCAAAACAGCTTAACAATAGAGAGAATGGTGCTCTCTGCTTTTGACGAAAGGAGAAATAAATACCTTGAAGAACATCCCAGTGCGGGAAAAGATCCTAAGAAAACTGGAGGACCTATATACAGGAGAGTAAACGGAAAGTGGATGAGAGAACTCATCCTTTATGACAAAGAAGAAATAAGGCGAATCTGGCGCCAAGCTAATAATGGTGACGATGCAACGGCTGGTCTGACTCACATGATGATCTGGCATTCCAATTTGAATGATGCAACTTATCAGAGGACAAGAGCTCTTGTTCGCACCGGAATGGATCCCAGGATGTGCTCTCTGATGCAAGGTTCAACTCTCCCTAGGAGGTCTGGAGCCGCAGGTGCTGCAGTCAAAGGAGTTGGAACAATGGTGATGGAATTGGTCAGAATGATCAAACGTGGGATCAATGATCGGAACTTCTGGAGGGGTGAGAATGGACGAAAAACAAGAATTGCTTATGAAAGAATGTGCAACATTCTCAAAGGGAAATTTCAAACTGCTGCACAAAAAGCAATGATGGATCAAGTGAGAGAGAGCCGGAACCCAGGGAATGCTGAGTTCGAAGATCTCACTTTTCTAGCACGGTCTGCACTCATATTGAGAGGGTCGGTTGCTCACAAGTCCTGCCTGCCTGCCTGTGTGTATGGGCCTGCCGTAGCCAGTGGGTACGACTTTGAAAGGGAGGGATACTCTCTAGTCGGAATAGACCCTTTCAGACTGCTTCAAAACAGCCAAGTGTACAGCCTAATCAGACCAAATGAGAATCCAGCACACAAGAGTCAACTGGTGTGGATGGCATGCCATTCTGCCGCATTTGAAGATCTAAGAGTATTAAGCTTCATCAAAGGGACGAAGGTGCTCCCAAGAGGGAAGCTTTCCACTAGAGGAGTTCAAATTGCTTCCAATGAAAATATGGAGACTATGGAATCAAGTACACTTGAACTGAGAAGCAGGTACTGGGCCATAAGGACCAGAAGTGGAGGAAACACCAATCAACAGAGGGCATCTGCGGGCCAAATCAGCATACAACCTACGTTCTCAGTACAGAGAAATCTCCCTTTTGACAGAACAACCATTATGGCAGCATTCAATGGGAATACAGAGGGGAGAACATCTGACATGAGGACCGAAATCATAAGGATGATGGAAAGTGCAAGACCAGAAGATGTGTCTTTCCAGGGGCGGGGAGTCTTCGAGCTCTCGGACGAAAAGGCAGCGAGCCCGATCGTGCCTTCCTTTGACATGAGTAATGAAGGATCTTATTTCTTCGGAGACAATGCAGAGGAGTACGACAAT

GGTGATCAGAATCCTCAAATTGCGGCACATGTCATAAGTGAGGCCAGCAGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAGGATACTACACCATGAGCAACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTATGCCCAAGTCACCTTCTGTTCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGCCTCTGCCTAAAGTCCCCCGGTAGATTCGAGAGAATCTTACTCAGAGCTGCAAATACCCACAGTTCCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGCTTCGGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTTGGCTTACTCAAACTCTGA <210> SEQ ID NO 10 <211>LENGTH: 922 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Human Surfactant Protein D collagen-like domain fused to H. influenza NP protein and the extra-cellular domain of human CD40L (minus stalk region (Non- Stalk (NST)))MLLFLLSALVLLTQPLGYLEAEMKTYSHRTTPSACTLVMCSSVESGLPGRDGRDGREGPRGEKGDPGLPGAAGQAGMPGQAGPVGPKGDNGSVGEPGPKGDTGPSMASQGTKRSYEQMETDGERQNATEIRASVGKMIGGIGREYIQMCTELKLSDYEGRLIQNSLTIERMVLSAFDERRNKYLEEHPSAGKDPKKTGGPIYRRVNGKWMRELILYDKEEIRRIWRQANNGDDATAGLTHMMIWHSNLNDATYQRTRALVRTGMDPRMCSLMQGSTLPRRSGAAGAAVKGVGTMVMELVRMIKRGINDRNFWRGENGRKTRIAYERMCNILKGKFQTAAQKAMMDQVRESRNPGNAEFEDLTFLARSALILRGSVAHKSCLPACVYGPAVASGYDFEREGYSLVGIDPFRLLQNSQVYSLIRPNENPAHKSQLVWMACHSAAFEDLRVLSFIKGTKVLPRGKLSTRGVQIASNENMETMESSTLELRSRYWAIRTRSGGNTNQQRASAGQISIQPTFSVQRNLPFDRTTIMAAFNGNTEGRTSDMRTEIIRMMESARPEDVSFQGRGVFELSDEKAASPIVPSFDMSNEGSYFFGDNAEEYDNGPPGPPGVPGPAGREGPLGKQGNIGPQGKPGPKGEAGPKGEVGAPGMQGSAGARGLAGPKGERGVPGERGVPGNTGAAGSAGAMGPQGSPGARGPPGLKGDKGIPGDKGAKGESGLPDVASLRQQVEALQGQVQHLQAAFSQYKKVELFPNGQSVGEKIFKTAGFVKPFTEAQGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL tpa-muACRP30-CSP1-muACRP30-muCD40L <210> SEQ ID NO 11 <211>LENGTH: 1929 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Human TPA signal sequence fused tomurine ACRP30 fused to P. yoelii CSP-1 and the extracellulardomain of murine CD40L Underlined sequence: Human TPA sequenceItalicized/bolded sequence: Murine ACRP30 sequenceNon-italicized/bolded sequence: P. yoelii CSP-1 sequenceItalicized sequence: Murine CD40L sequence (extracellular domain)ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCC CAGC

AAAATATACAATCGAAATATAGTCAACAGATTACTTGGCGATGCTCTCAACGGAAAACCAGAAGAAAAAAAAGATGATCCCCCAAAAGATGGCAACAAAGATGATCTTCCAAAAGAAGAAAAAAAAGATGATCTTCCAAAAGAAGAAAAAAAAGATGATCCCCCAAAAGATCCTAAAAAAGATGATCCACCAAAAGAGGCTCAAAATAAATTGAATCAACCAGTAGTGGCAGATGAAAATGTAGATCAAGGGCCAGGAGCACCACAAGGGCCAGGAGCACCACAAGGACCAGGAGCACCACAGGGTCCAGGAGCACCACAAGGACCAGGAGCACCACAAGGACCAGGAGCACCACAAGGTCCAGGAGCACCACAGGGTCCAGGAGCACCACAGGGTCCAGGAGCACCACAAGGACCAGGAGCACCACAGGGGCCAGGAGCACCACAAGGACCAGGAGCACCACAAGGACCAGGAGCACCACAGGGGCCAGGAGCACCACAAGGGCCAGGAGCACCACAAGAACCACCCCAACAACCACCCCAACAACCACCACAACAGCCACCACAACAGCCACCACAACAGCCACCACAACAGCCACCACAACAACCACGCCCACAGCCAGATGGTAATAACAACAATAACAATAATAATGGTAATAATAATGAAGATTCTTATGTCCCAAGCGCGGAACAAATACTAGAATTTGTTAAACAGATAAGTAGTCAACTCACAGAGGAATGGTCTCAATGTAGTGTAACCTGTGGTTCTGGTGTAAGAGTTAGAAAACGAAAAAATGTAAACAAGCAACCAGAAAATTTGACCTTAGAGGATATTGATACTGAAATTTGTAAAATGGATAAATGTTCAAGTATATTTAATATTGTAAGCAATTCATTAGGATTTGTAATATTATTAGTATTAGTATTCTTTAAT

CATAGAAGATTGGATAAGGTCGAAGAGGAAGTAAACCTTCATGAAGATTTTGTATTCATAAAAAAGCTAAAGAGATGCAACAAAGGAGAAGGATCTTTATCCTTGCTGAACTGTGAGGAGATGAGAAGGCAATTTGAAGACCTTGTCAAGGATATAACGTTAAACAAAGAAGAGAAAAAAGAAAACAGCTTTGAAATGCAAAGAGGTGATGAGGATCCTCAAATTGCAGCACACGTTGTAAGCGAAGCCAACAGTAATGCAGCATCCGTTCTACAGTGGGCCAAGAAAGGATATTATACCATGAAAAGCAACTTGGTAATGCTTGAAAATGGGAAACAGCTGACGGTTAAAAGAGAAGGACTCTATTATGTCTACACTCAAGTCACCTTCTGCTCTAATCGGGAGCCTTCGAGTCAACGCCCATTCATCGTCGGCCTCTGGCTGAAGCCCAGCATTGGATCTGAGAGAATCTTACTCAAGGCGGCAAATACCCACAGTTCCTCCCAGCTTTGCGAGCAGCAGTCTGTTCACTTGGGCGGAGTGTTTGAATTACAAGCTGGTGCTTCTGTGTTTGTCAACGTGACTGAAGCAAGCCAAGTGATCCACAGAGTTGGCTTCTCATCTTTTGGCTTACTCAAACTCTGA <210> SEQ ID NO 12 <211>LENGTH: 642 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Human TPA signal sequence fused tomurine ACRP30 fused to P. yoelii CSP-1 and the extracellulardomain of murine CD40LMDAMKRGLCCVLLLCGAVFVSPSEDDVTTTEELAPALVPPPKGTCAGWMAGIPGHPGHNGTPGRDGKIYNRNIVNRLLGDALNGKPEEKKDDPPKDGNKDDLPKEEKKDDLPKEEKKDDPPKDPKKDDPPKEAQNKLNQPVVADENVDQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQGPGAPQEPPQQPPQQPPQQPPQQPPQQPPQQPPQQPRPQPDGNNNNNNNNGNNNEDSYVPSAEQILEFVKQISSQLTEEWSQCSVTCGSGVRVRKRKNVNKQPENLTLEDIDTEICKMDKCSSIFNIVSNSLGFVILLVLVFFNRDGTPGEKGEKGDAGLLGPKGETGDVGMTGAEGPRGFPGTPGRKGEPGEAAHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSIGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKLmuSP-D-Gag-muSP-D-muRANKL <210> SEQ ID NO 13 <211> LENGTH: 3006 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to HIV-1 Gag and the extra cellular domainof murine RANKLItalicized/bolded sequence: Murine SP-D sequence (collagen- like domain)Non-italicized/bolded sequence: HIV-1 Gag sequence (shifted10aa along SP-D)Italicized sequence: Murine RANKL sequence (extracellular domain)

ATGGGAGCCAGGGCCAGCGTGCTGTCTGGGGGCGAGCTGGACAGGTGGGAGAAGATTAGGCTGAGGCCCGGAGGAAAGAAGAAGTACAAACTGAAACACATCGTGTGGGCCTCCCGGGAGCTGGAACGGTTCGCCGTGAATCCTGGGCTGCTGGAGACCTCTGAGGGCTGCAGACAGATCCTGGGACAGCTGCAGCCTAGCCTGCAGACCGGAAGCGAGGAGCTGAGGTCTCTGTACAACACCGTGGCCACACTGTACTGCGTGCACCAGCGGATTGAGGTGAAGGATACCAAGGAAGCCCTGGAGAAGATTGAGGAAGAGCAGAATAAGTCCAAGAAGAAAGCCCAGCAGGCCGCCGCCGACACAGGAAATAGCTCCCAGGTGTCTCAGAACTACCCCATCGTGCAGAACCTGCAGGGACAGATGGTGCACCAGGCCATCAGCCCCCGGACCCTGAACGCCTGGGTGAAGGTGGTGGAAGAGAAAGCCTTCAGCCCAGAAGTGATCCCCATGTTCAGCGCCCTGAGCGAAGGGGCCACCCCACAGGACCTGAATACAATGCTGAATACAGTGGGCGGCCACCAGGCCGCCATGCAGATGCTGAAGGAGACCATTAACGAGGAGGCCGCCGAGTGGGATAGGCTGCACCCAGTGCACGCCGGGCCCATCGCCCCAGGGCAGATGAGGGAGCCACGGGGCTCTGACATCGCCGGCACCACCTCTACCCTGCAGGAGCAGATCGGCTGGATGACCAATAACCCACCTATTCCCGTGGGAGAAATCTACAAAAGGTGGATTATCCTGGGGCTGAACAAGATCGTGAGAATGTACTCCCCAACATCCATTCTGGACATCCGGCAGGGCCCAAAGGAACCCTTTAGAGACTACGTGGATAGGTTCTACAAAACCCTGCGCGCCGAGCAGGCCTCCCAGGAGGTGAAGAACTGGATGACCGAGACACTGCTGGTGCAGAATGCCAACCCAGACTGTAAGACCATTCTGAAGGCCCTGGGACCAGCCGCCACCCTGGAGGAAATGATGACAGCCTGCCAGGGGGTGGGCGGACCCGGCCACAAGGCCCGCGTGCTGGCCGAGGCCATGTCCCAGGTGACAAATTCCGCCACCATCATGATGCAGCGCGGAAATTTTCGGAATCAGCGCAAAACAGTGAAATGCTTCAATTGCGGGAAGGAGGGCCACATCGCCAAGAATTGCAGAGCCCCAAGGAAGAAGGGCTGCTGGAAGTGCGGAAAGGAGGGCCACCAGATGAAGGACTGCACAGAGCGCCAGGCCAATTTCCTGGGCAAGATCTGGCCATCCCACAAGGGGCGGCCTGGAAACTTCCTGCAGAGCCGGCCCGAACCCACAGCCCCCCCTGAAGAATCCTTCCGGTTCGGAGAGGAAACAACCACACCCAGCCAGAAGCAGGAGCCTATCGACAAGGAACTGTACCCACTGGCCAGCCTGAGAAGCCTGTTCGGCAACGATCCAAGCAGCCAG

CGAGCGCAGATGGATCCTAACAGAATATCAGAAGACAGCACTCACTGCTTTTATAGAATCCTGAGACTCCATGAAAACGCAGATTTGCAGGACTCGACTCTGGAGAGTGAAGACACACTACCTGACTCCTGCAGGAGGATGAAACAAGCCTTTCAGGGGGCCGTGCAGAAGGAACTGCAACACATTGTGGGGCCACAGCGCTTCTCAGGAGCTCCAGCTATGATGGAAGGCTCATGGTTGGATGTGGCCCAGCGAGGCAAGCCTGAGGCCCAGCCATTTGCACACCTCACCATCAATGCTGCCAGCATCCCATCGGGTTCCCATAAAGTCACTCTGTCCTCTTGGTACCACGATCGAGGCTGGGCCAAGATCTCTAACATGACGTTAAGCAACGGAAAACTAAGGGTTAACCAAGATGGCTTCTATTACCTGTACGCCAACATTTGCTTTCGGCATCATGAAACATCGGGAAGCGTACCTACAGACTATCTTCAGCTGATGGTGTATGTCGTTAAAACCAGCATCAAAATCCCAAGTTCTCATAACCTGATGAAAGGAGGGAGCACGAAAAACTGGTCGGGCAATTCTGAATTCCACTTTTATTCCATAAATGTTGGGGGATTTTTCAAGCTCCGAGCTGGTGAAGAAATTAGCATTCAGGTGTCCAACCCTTCCCTGCTGGATCCGGATCAAGATGCGACGTACTTTGGGGCTTTCAAAGTTCAGGACATAGACTGA <210> SEQ ID NO 14 <211> LENGTH: 1001 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to HIV-1 Gag and the extracellular domain ofmurine RANKLMLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDGRDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERGLSGPPGLPGIPGPMGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEVKDTKEALEKIEEEQNKSKKKAQQAAADTGNSSQVSQNYPIVQNLQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRLHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTNNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPAATLEEMMTACQGVGGPGHKARVLAEAMSQVTNSATIMMQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGNDPSSQAGKEGPSGKQGNIGPQGKPGPKGEAGPKGEVGAPGMQGSTGAKGSTGPKGERGAPGVQGAPGNAGAAGPAGPAGPQGAPGSRGPPGLKGDRGVPGDRGIKGESGLPDSAALRQQMEALKGKLQRLEVAFSHYQKAALFPDGRAQMDPNRISEDSTHCFYRILRLHENADLQDSTLESEDTLPDSCRRMKQAFQGAVQKELQHIVGPQRFSGAPAMMEGSWLDVAQRGKPEAQPFAHLTINAASIPSGSHKVTLSSWYHDRGWAKISNMTLSNGKLRVNQDGFYYLYANICFRHHETSGSVPTDYLQLMVYVVKTSIKIPSSHNLMKGGSTKNWSGNSEFHFYSINVGGFFKLRAGEEISIQVSNPSLLDPDQDATYFGAFKVQDID huSP-D-WT1-huSP-D-huCD40L <210> SEQ ID NO 15<211> LENGTH: 1986 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223>OTHER INFORMATION: Human Surfactant Protein D collagen-like domain fused to human WT1 and the extracellular domainof human CD40LItalicized/bolded sequence: Human SP-D sequence (collagen- like domain)Non-italicized/bolded sequence: Human WT1 sequenceItalicized sequence: Human CD40L sequence (extracellular domain)

ATGGGCCATCATCATCATCATCATCATCATCATCACAGCAGCGGCCATATCGAAGGTCGTCATATGCGACGTGTGCCTGGAGTAGCCCCGACTCTTGTACGGTCGGCATCTGAGACCAGTGAGAAACGCCCCTTCATGTGTGCTTACCCAGGCTGCAATAAGAGATATTTTAAGCTGTCCCACTTACAGATGCACAGCAGGAAGCACACTGGTGAGAAACCATACCAGTGTGACTTCAAGGACTGTGAACGAAGGTTTTTTCGTTCAGACCAGCTCAAAAGACACCAAAGGAGACATACAGGTGTGAAACCATTCCAGTGTAAAACTTGTCAGCGAAAGTTCTCCCGGTCCGACCACCTGAAGACCCACACCAGGACTCATACAGGTGAAAAGCCCTTCAGCTGTCGGTGGCCAAGTTGTCAGAAAAAGTTTGCCCGGTCAGATGAATTAGTCCGCCATCACAACATGCATCAGAGAAACATGACCAAACTCCAGCTGGCGCTT

CATAGAAGGTTGGACAAGATAGAAGATGAAAGGAATCTTCATGAAGATTTTGTATTCATGAAAACGATACAGAGATGCAACACAGGAGAAAGATCCTTATCCTTACTGAACTGTGAGGAGATTAAAAGCCAGTTTGAAGGCTTTGTGAAGGATATAATGTTAAACAAAGAGGAGACGAAGAAAGAAAACAGCTTTGAAATGCAAAAAGGTGATCAGAATCCTCAAATTGCGGCACATGTCATAAGTGAGGCCAGCAGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAGGATACTACACCATGAGCAACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTATGCCCAAGTCACCTTCTGTTCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGCCTCTGCCTAAAGTCCCCCGGTAGATTCGAGAGAATCTTACTCAGAGCTGCAAATACCCACAGTTCCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGCTTCGGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTTGGCTTACTCAAACTCTGA <210> SEQ ID NO 16 <211>LENGTH: 661 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Human Surfactant Protein D collagen-like domain fused to human WT1 and the extracellular domainof human CD40LMLLFLLSALVLLTQPLGYLEAEMKTYSHRTTPSACTLVMCSSVESGLPGRDGRDGREGPRGEKGDPGLPGAAGQAGMPGQAGPVGPKGDNGSVGEPGPKGDTGPSMGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLALGPPGPPGVPGPAGREGPLGKQGNIGPQGKPGPKGEAGPKGEVGAPGMQGSAGARGLAGPKGERGVPGERGVPGNTGAAGSAGAMGPQGSPGARGPPGLKGDKGIPGDKGAKGESGLPDVASLRQQVEALQGQVQHLQAAFSQYKKVELFPNGQSVGEKIFKTAGFVKPFTEAQHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL muSP-D-MAGE-A3-muSP-D-muBAFF <210> SEQ ID NO 17 <211>LENGTH: 2394 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to murine MAGE-A3 and the extracellular domain of murine BAFF. Deleted 20aa of SP-D sequence.Italicized/bolded sequence: Murine SP-D sequence (collagen-like domain) (deleted 20aa)Non-italicized/bolded sequence: Murine MAGE-A3 sequenceItalicized sequence: Murine BAFF sequence (extracellular domain)

ATGGCTGATTCCCATAACACCCAATACTGCAACCTCGAAGAGAGTGCTCAGGCCCAACAGGAATTAGACAATGACCAGGAGACCATGGAGACATCAGAGGAGGAGGAAGATACCACCACCTCAAATAAAGTGTATGGCAGTGCAATACCAAGTCCTCCCCAGAGTCCTCAGAGAGCCTACTCTCCCTGTGTGGCACTGGCCTCCATCCCTGATAGCCCATCTGAGGAAGCTTCCATCAAAGGATCAGAGGGCCTGGAAGACCCACTTCATTTGTTGCACAATGCACAGAACACAAAGGTGTATGACTTGGTGGACTTTCTGGTTTTAAACTATCAAATGAAGGCATTCACTACCAAAGCAGAAATGTTGGAAAATATTGGTAGAGAGTATGAGGAGTACTACCCTCTGATCTTTAGTGAGGCCTCTGAGTGCTTGAAGATGGTCTTTGGCCTTGACATGGTAGAAGTGGACTCCTCTGTCCACACCTATATGCTTGTCACTGCCCTGGGGATCACCTATGATGGCATGATGACTGATGTCCAGGGTATGCCCAAGACAGGTATCCTCATAGCTGTACTGAGTGTCATTTTCATGAAGGGAAACTATGTCAGTGAGGAGATTATCTGGGAAATGCTGAATAACATAGGGTTGTGTGGTGGGAGGGATCCTTACATACATAAAGACCCCAGGAAGCTCATCTCTGAGGAGTTTGTGCAGGAAGGGTACCTGGAATACAGGCAGGTGCCCAATAGTGATCCCCCTAGCTATGGGTTCCTGTGGGGCCCAAGGGCTTTTGCAGAAACCAGCAAAATGAAAGTCTTACAGTTCTTTGCCAGCATTAATAAGACTCATCCCAGAGCCTACCCTGAAAAGTATGCAGAGGCTTTACAAGATGAGATAGACAGGACCAAGACCTGGATCTTGAACAGATGCTCCAACTCCTCTGACCTACACACATTC

CAGTTGGCTGCCTTGCAAGCAGACCTGATGAACCTGCGCATGGAGCTGCAGAGCTACCGAGGTTCAGCAACACCAGCCGCCGCGGGTGCTCCAGAGTTGACCGCTGGAGTCAAACTCCTGACACCGGCAGCTCCTCGACCCCACAACTCCAGCCGCGGCCACAGGAACAGACGCGCTTTCCAGGGACCAGAGGAAACAGAACAAGATGTAGACCTCTCAGCTCCTCCTGCACCATGCCTGCCTGGATGCCGCCATTCTCAACATGATGATAATGGAATGAACCTCAGAAACATCATTCAAGACTGTCTGCAGCTGATTGCAGACAGCGACACGCCGACTATACGAAAAGGAACTTACACATTTGTTCCATGGCTTCTCAGCTTTAAAAGAGGAAATGCCTTGGAGGAGAAAGAGAACAAAATAGTGGTGAGGCAAACAGGCTATTTCTTCATCTACAGCCAGGTTCTATACACGGACCCCATCTTTGCTATGGGTCATGTCATCCAGAGGAAGAAAGTACACGTCTTTGGGGACGAGCTGAGCCTGGTGACCCTGTTCCGATGTATTCAGAATATGCCCAAAACACTGCCCAACAATTCCTGCTACTCGGCTGGCATCGCGAGGCTGGAAGAAGGAGATGAGATTCAGCTTGCAATTCCTCGGGAGAATGCACAGATTTCACGCAACGGAGACGACACCTTCTTTGGTGCCCTAAAACTGCTGTAA <210>SEQ ID NO 18 <211> LENGTH: 797 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Murine Surfactant Protein D collagen-like domain fused to murine MAGE-A3 and the extracellulardomain of murine BAFF. Deleted 20aa of SP-D sequence.MLPFLSMLVLLVQPLGNLGAEMKSLSQRSVPNTCTLVMCSPTENGLPGRDGRDGREGPRGEKGDPGLPGPMGLSGLQGPTGPVGPKGENGSAGEPGPKGERGLSGMADSHNTQYCNLEESAQAQQELDNDQETMETSEEEEDTTTSNKVYGSAIPSPPQSPQRAYSPCVALASIPDSPSEEASIKGSEGLEDPLHLLHNAQNTKVYDLVDFLVLNYQMKAFTTKAEMLENIGREYEEYYPLIFSEASECLKMVFGLDMVEVDSSVHTYMLVTALGITYDGMMTDVQGMPKTGILIAVLSVIFMKGNYVSEEIIWEMLNNIGLCGGRDPYIHKDPRKLISEEFVQEGYLEYRQVPNSDPPSYGFLWGPRAFAETSKMKVLQFFASINKTHPRAYPEKYAEALQDEIDRTKTWILNRCSNSSDLHTFGNIGPQGKPGPKGEAGPKGEVGAPGMQGSTGAKGSTGPKGERGAPGVQGAPGNAGAAGPAGPAGPQGAPGSRGPPGLKGDRGVPGDRGIKGESGLPDSAALRQQMEALKGKLQRLEVAFSHYQKAALFPDGQLAALQADLMNLRMELQSYRGSATPAAAGAPELTAGVKLLTPAAPRPHNSSRGHRNRRAFQGPEETEQDVDLSAPPAPCLPGCRHSQHDDNGMNLRNIIQDCLQLIADSDTPTIRKGTYTFVPWLLSFKRGNALEEKENKIVVRQTGYFFIYSQVLYTDPIFAMGHVIQRKKVHVFGDELSLVTLFRCIQNMPKTLPNNSCYSAGIARLEEGDEIQLAIPRENAQISRNGDDTFFGALKLL

1. A composition comprising: (a) a multimerization scaffold, operativelylinked to (b) a plurality of a TNFSF receptor binder where two or morecomplete TNFSF receptors must be bound in order to activate a cell,operatively linked to (c) one or more antigens recognized by the immunesystem (d) where the composition does not contain portions of avidin orstreptavidin.
 2. The composition of claim 1, wherein the multimerizationscaffold (a) and the plurality of TNFSF receptor binder (b) do notcontain xenogenic portions.
 3. The composition of claim 1, wherein themultimerization scaffold is comprised of a protein selected from thecollectin or C1q superfamilies from which the natural C-terminal domainhas been removed and replaced by an operatively linked TNFSF receptorbinder.
 4. The composition of claim 1, wherein the multimerizationscaffold is comprised of a dimerizing component operatively linked to anantigen which is operatively linked to a trimerizing component which isoperatively linked to a TNFSF receptor binder, where the TNFSF binder isnot OX40L.
 5. The composition of claim 4, wherein the dimerizingcomponent is the Fc portion of an immunoglobulin.
 6. The composition ofclaim 4, wherein trimerizing domain is selected from the groupcomprising coiled-coil region of yeast GCN4 isoleucine variant, TRAF2,thrombospondin 1, Matrilin-4, cubilin, or the neck region of surfactantprotein D.
 7. The composition of claim 1, wherein the multimerizationscaffold is prepared by chemical methods.
 8. The composition of claim 1,wherein the TNFSF receptor binder is comprised of an extracellulardomain selected from a TNFSF proteins.
 9. The composition of claim 1,wherein the TNFSF receptor binder is comprised of the protein sequenceof the binding site of an antibody that binds to a TNFSF receptor. 10.An antigen-presenting cell (APC) that has been treated with thecomposition of claim 1 such that the treated APC can be administered toa subject as a vaccine or immunotherapy, wherein the APC is selectedfrom the group of dendritic cells, monocytes, macrophages, or B cells.11. An antigen-presenting cell (APC) of claim 10 that can be used invitro to generate immune cells and the generated immune cells areadministered to a subject as a vaccine or immunotherapy, wherein theimmune cells are selected from CD4+ T cells, CD8+ T cells, or B cells.12. The composition of claim 1 administered to a subject as a vaccine orimmunotherapy to elicit an immune response against the antigen in asubject in need of this immune response.
 13. The APC of claim 10administered to a subject as a vaccine or immunotherapy to elicit animmune response against the antigen in a subject in need of this immuneresponse.
 14. The immune cells of claim 11 administered to a subject asa treatment for an infectious agent or cancer in a subject in need ofthese immune cells. 15.-20. (canceled)
 21. The vaccine or immunotherapyof claim 12 encoded by a nucleic acid sequence that is delivered to asubject as DNA or RNA either alone or mixed with polymers to enhance theexpression of protein from the nucleic acid sequence in vivo. 22.(canceled)
 23. The vaccine or immunotherapy of claim 12 encoded by anucleic acid sequence that is delivered to a subject using a viralvector selected from but not limited to adenoviruses, poxviruses,alphaviruses, arenaviruses, flaviruses, rhabdoviruses, retroviruses,lentiviruses, herpesviruses, paramyxoviruses, and picornaviruses.24.-29. (canceled)
 30. A kit comprising APCs according to claim 10 thatcan be used in vitro to generate immune cells and wherein the generatedimmune cells are administered to a subject as a vaccine or immunotherapyand where the immune cells are selected from CD4+ T cells, CD8+ T cells,or B cells.
 31. A fusion protein comprising a multimerization scaffold,at least one TNFSF receptor binder, and at least one antigen.
 32. Thefusion protein of claim 31, wherein the antigen is positionedC-terminally from the multimerization scaffold or within themultimerization scaffold. 33.-36. (canceled)
 37. The fusion protein ofclaim 31, comprising the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,16, or
 18. 38.-45. (canceled)